Observations on the role of smooth endoplasmic reticulum in glucocorticoid-induced hepatic glycogen deposition

We have studied by quantitative electron microscopy the relationship of specific hepatic cellular organelles to glycogen synthesis using dexamethasone, a potent synthetic glucocorticoid, to induce glycogen deposition in livers of adrenalectomized rats. Chemical and ultrastructural glycogen determinations revealed that the livers of fasted adrenalectomized rats had very low glycogen levels. Dexamethasone caused a time-related increase in hepatic glycogen which was the result of increases in the number of hepatocytes depositing glycogen and the amount of glycogen in each cell. The surface density of smooth endoplasmic reticulum (SER) in centrilobular and periportal hepatocytes also increased after treatment with dexamethasone; this increase preceded glycogen deposition. The newly deposited glycogen was spatially associated with membranes of SER, and a continued increase in SER surface density was correlated temporally with the increasing glycogen volume density. In both centrilobular and periportal hepatocytes, the suface density of rough endoplasmic reticulum (RER) initially decreased after dexamethasone administration but later increased. These data support the hypothesis that dexamethasone-induced enhancement of SER is functionally associated with the increase in glycogen, and that although the initial increase in SER may occur through transformation of RER to SER, later increases in SER require synthesis of new membranes.     

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with -amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca²⁺. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.Abbreviations SER smooth endoplasmic reticulum - RER rough endoplasmic reticulum - PMS post mitochondrial supernatant - MES 2-(N-morpholino) ethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Comparative ultrastructure of Clara cells in neonatal and older cattle

Calf lungs were fixed with glutaraldehyde and examined by scanning (SEM) and transmission (TEM) electron microscopy to compare the ultrastructure of Clara cells in terminal bronchioles of neonatal calves and older cattle. In the neonatal calf, SEM revealed numerous smooth-surfaced Clara cells protruding above a similar number of ciliated cells, whereas in older animals the surface of Clara cells was lobulated. Thin sections examined by TEM revealed numerous cuboidal to columnar Clara cells with indented nuclei and a pale cytoplasm filled with faintly granular glycogen in the neonatal calf. Some cells were characterized by apical dense and/or pale membrane-bound granules or secretory droplets. Many cells had an apical tubular network of cisternae that were partly smooth and partly decorated with ribosomes. Ultrastructural comparison of Clara cells in a 2-day-old calf with those of 14- and 19-day-old, 4- and 5. 5-month-old, and 3.5-year-old cattle revealed a striking reduction in the amount of glycogen per cell after 14 days. The number of cells with apical granules was small at all ages, and the density of the secretory granules varied greatly in different cells. A variable amount of smooth endoplasmic reticulum (SER) was present but was less prominent than cisternae of ribosomal endoplasmic reticulum (RER). In older cattle, the limited amount of SER compared to the RER and secretory granules suggests that bovine Clara cells are more likely to be secretory than detoxifying.     

The Ultrastructure of Circulating Hemolymph Cells of the Marine Snail Cerithidea californica (Gastropoda: Prosobranchiata)

Two morphologically distinct blood cell-types, the granulocyte and hyalinocyte, are found in the hemolymph circulation of the marine prosobranch Cerithidea californica. Granulocytes, measuring 12.7 µ (9.0–15.0 µ) in diameter, possess well-defined ectoplasmic and endoplasmic regions of the cytoplasm, granules of moderate to heavy electron density, tubular rough endoplasmic reticulum (RER), short vesicles of smooth endoplasmic reticulum (SER), and a large cytoplasm to nucleus ratio. Two morphological variants of this cell-type are distinguished depending upon the presence or absence of dense granules or RER. Hyalinocytes, measuring 5.3 µ (4.0–8.0 µ) in diameter, are distinguished from gran ulocytes by possessing a smaller cytoplasm to nucleus ratio and a general lack of dense cytoplasmic granules and SER.     

Zonal changes in the rat liver following an acute dose of phenobarbitone: An ultrastructural,morphometric and biochemical correlation

Alterations in the liver of rats 6 h after a dose of phenobarbitone have been studied by subcellular fractionation, conventional electron microscopy and morphometric analysis. The area immediately surrounding the central vein was the only area to undergo any alterations. There was a morphometrically measurable but not observable cellular hypertrophy of 71% whilst the hepatocyte complement of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) was increased by 72% and 93% respectively. The increases in RER and SER were not apparent by observation and it is assumed that they have been diluted by the cell hypertrophy to 1% and 22% which must be below the threshold for detection by subjective observation. Following subcellular fractionation and measurement of microsomal protein, there was no significant difference in the level of microsomes isolated from control or treated rats. Therefore, the morphometrically measured increase in RER and SER would appear to be restricted to a relatively small population of hepatocytes adjacent to the central vein. Such an increase would represent only a small percentage of total microsomes in a homogenate and would almost certainly be masked by variation in animals and techniques. Disruption of RER was also observed in hepatocytes that would proliferate their SER should phenobarbitone treatment have been continued. Therefore this RER disruption would seem in no way to interfere with the process of membrane and enzyme synthesis.     

CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL : II. Effects of Phenobarbital on Rat Hepatocytes

The changes occurring in rat hepatocytes during a 5 day period of treatment with phenobarbital were determined by morphometric and biochemical methods, particular attention being paid to the endoplasmic reticulum. The hepatocytic cytoplasm played an overwhelming part in the liver hypertrophy, while the hepatocytic nuclei contributed to only a moderate extent. The endoplasmic reticulum accounted for more than half of the increase in cytoplasmic volume. The increase in the volume and number of hepatocytic nuclei in the course of phenobarbital treatment was associated with changes in the ploidy pattern. Until the 2nd day of treatment both the rough-surfaced endoplasmic reticulum (RER) and the smooth-surfaced endoplasmic reticulum (SER) participated in the increase in volume and surface of the whole endoplasmic reticulum (ER). Subsequently, the values for RER fell again to control levels, whereas those for SER continued to increase, with the result that by the 5th day of treatment the SER constituted the dominant cytoplasmic element. The specific volume of mitochondria and microbodies (peroxisomes) remained constant throughout the duration of the experiment, while that of the dense bodies increased. The specific number of mitochondria and microbodies displayed a significant increase, associated with a decrease in their mean volume. The phenobarbital-induced increase in the phospholipid and cytochrome P-450 content of the microsomes, as well as in the activities of microsomal reduced nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase and N-demethylase, was correlated with the morphometric data on the endoplasmic reticulum.     

Effect of lysine and threonine deficient and supplement rice diets on lipids of hepatic smooth and rough endoplasmic reticulum of rats

Effect of feeding lysine and threonine deficient and supplemented rice diets to rats for fifteen days has been studied on the lipids of hepatic smooth (SER) and rough endoplasmic reticulum. The deficient diet reduced protein and phospholipids of SER where as it increased triglycerides contents of both SER and RER compared to rats fed the supplemented diet.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

The ultrastructure of hemocytes inDactylopius confusus (Cockerell), and the role of granulocytes in the synthesis of cochineal dye

Summary The ultrastructural study of free circulating hemocytes in the adult cochineal scale,Dactylopius confusus (Cockerell), demonstrated five cell types: prohemocytes, typical granulocytes (T-granulocytes), oenocytoids, plasmatocytes, and granulocytes with modified sub-cellular structure to perform a special synthetic and secretory function, which we refer to as modified granulocytes (M-granulocytes). Prohemocytes showed undifferentiated sub-cellular structure of the basic stem cell type (i.e., high cytoplasmic density with numerous ribosomes, centrally located large nucleus with a distinct nucleolus, and poorly developed endoplasmic reticulum). The commonly observed typical granulocytes (T-granulocytes) had several smooth endoplasmic reticulum (SER) with dilated cisternae and many SER-derived membrane bounded granules of different sizes and electron density. Oenocytoids were identified by the presence of many crystals, RER-originated fine secretory granules, and an eccentric nucleus. Plasmatocytes were easily characterized by their variable shapes and irregular outline with pseudopodia-like cytoplasmic extensions, possession of an elongated lobed nucleus, multivesicular bodies, RER-derived membrane bounded, electron-dense, lysosomelike vacuoles, well-developed SER cisternae, and numerous pinocytic and SER-originated vesicles of different sizes along the peripheral region. M-granulocytes comprised the largest proportion of hemocytes in all samples observed. M-granulocytes were distinguished not only by the presence of membrane bounded granules of different sizes and electron density, but by the possession of large nuclei with distinct nucleoli, many mitochondria, and a highly developed network of rough endoplasmic reticulum (RER). M-granulocytes had abundant, rosette-shaped, RER-derived chains of fine secretory granules, which accumulated in the cytoplasm and vacuoles, and were ultimately deposited into the hemolymph by exocytosis. These fine granules gave a positive result with periodic acid-Schiff (PAS) test. Based on RER-synthesized fine secretory granules (M-granulocytes), their ultimate deposition into hemolymph, the red pigmentation of hemolymph, positive PAS histochemical test of these granules, and the high population of these hemocytes, no such cell type has been described in previous studies in insects. The sub-cellular structure of the granulocyte in this insect has been modified to perform a special synthetic and secretory function (i.e., possibly the synthesis of the red pigment found in hemolymph, which has been the source of commercially important cochineal dye).Abbreviations EM electron microscope - ER endoplasmic reticulum - LM light microscopy - MVB multivesicular body - PAS periodic acid-Schiff - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum - SG secretory granules - TEM transmission electron microscopy - UA uranyl acetate     

Stereological analysis of hepatic fine structure in the Fischer 344 rat. Influence of sublobular location and animal age

Stereological analysis of hepatic fine structure in Fischer 344 male rats at 1, 6, 10, 16, 20, 25, and 30 mo of age revealed differences in the amounts and distributions of hepatocellular organelles as a function of sublobular location or animal age. Between 1 and 16 mo of age, both the centrolobular and periportal hepatocytes increased in volume by 65 and 35%, respectively. Subsequently, the cell volumes declined until the hepatocytes of 30-mo-old rats approached the size of those found in the youngest animals. Regardless of animal age, the centrolobular cells were consistently larger than the corresponding periportal hepatocytes. The cytoplasmic and ground substance compartments reflected similar changes in their volumes, although there was no significant alteration in the nuclear volume. The volumes of the mitochondrial and microbody compartments increased and decreased concomitant with the changes in average hepatocyte size. Both lobular zones in the 30-mo-old rats contained significantly smaller relative volumes of mitochondria than similar parenchyma in 16-mo-old animals. The volume density of the dense bodies (lysosomes) increased markedly in both lobular zones between 1 and 30 mo of age, confirming reports of an age-dependent increase in this organelle. The surface area of the endoplasmic reticulum in the centrolobular and periportal hepatocytes reached its maximum level in the 10-mo-old rats and subsequently declined to amounts which approximated those measured in the 1-mo-old animals. This age-related loss of intracellular membrane is attributable to a significant reduction in the surface area of the smooth-surfaced endoplasmic reticulum (SER) in animals beyond 16 mo of age. The amount of rough-surfaced endoplasmic reticulum (RER) in the periportal parenchymal cells was unaffected by aging, but the centrolobular hepatocytes of 30-mo-old animals contained 90% more RER than similar cells in the youngest rats. The centrolobular parenchyma contained more SER and the portal zones more RER throughout the age span studied. These quantitative data suggest that (a) certain hepatic fine structural parameters undergo marked changes as a function of animal age, (b) there exists a gradient in hepatocellular fine structure across the entire liver lobule, and (c) there are remarkable similarities in hepatocyte ultrastructure between very young and senescent animals, including cell size and the amount of SER.     

Morphology of the liver of the brook lamprey, Lampetra lamottenii before and during infection with the nematode, Truttaedacnitis stelmioides, hepatocytes, sinusoids, and perisinusoidal cells.

Routine light microscopy and transmission and scanning electron microscopy were used to describe and compare the livers of larval lampreys, Lampetra lamottenii before and during infection of the bile ducts by the nematode, Truttaedacnitis stelmioides. The hepatocytes of uninfected animals differ from other lamprey species in that they contain abundant glycogen, smooth endoplasmic reticulum (SER), and lipoprotein indicating that the liver may be involved in glucose metabolism. Infestation of the biliary tree by T. stelmioides coincides with alterations to the hepatocytes. These changes include dilation of the bile canaliculi, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and Golgi apparatus, swollen mitochondria in cells showing a high degree of hypertrophy, and an abundance of dense bodies. Following infection, the sinusoidal lumina became dilated and contain a moderate electron-dense precipitate, an abundance of melanomacrophages, lipocytes, and mononuclear cells. There is also a widening of the fenestrae of the sinusoidal endothelium following infection. Many of the changes in hepatocytes and sinusoids following parasite infections closely resemble those observed in hepatocytes in various pathologies and following experimental bile duct ligation and, therefore, are likely a consequence of increased biliary pressure due to bile duct obstruction.     

Fine structure of hepatocytes from fasted and fed rats.

The fine structure of hepatocytes from rats maintained on a controlled feeding schedule are described. Liver samples were processed for electron microscopy, histochemistry and chemical determinations of glycogen at precise time-intervals following a 30-hour fast and a 2-hour meal. Hepatocytes from 30-hour-fasted rats with extremely low hepatic glycogen levels were devoid of glycogen particles. Centrilobular cells showed areas of the cytoplasm rich in vesicles of smooth endoplasmic reticulum (SER) while periportal hepatocytes contained less extensive regions of SER. Soon after feeding the fasted rats, glycogen particles appeared in regions of the cell rich in SER. Centrilobular hepatocytes contained numerous glycogen areas which were infiltrated with tubules of SER, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the masses of glycogen. Throughout glycogen deposition each glycogen particle was closely associated with membranes of SER until maximum glycogen deposition was achieved 12 hours after initiation of feeding. At this point SER was reduced to the lowest amounts of the time-periods studied. During stages of glycogen depletion SER proliferated and reached the highest concentration measured in this study. Tubules of SER were present throughout the glycogen masses of centrilobular hepatocytes, whereas in periportal cells the organelle was restricted to the periphery of the glycogen masses. It is concluded that SER is associated with glycogen particles in rat hepatocytes during both deposition and depletion of glycogen.     

The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultrastructural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultrastructure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.     

Phenobarbital-induced alterations in the activities of the transport and hydrolytic components of the glucose-6-phosphatase system in smooth and rough subfractions of the rat hepatic endoplasmic reticulum

We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

OVARIAN STEROID CELLS : I. Differentiation of the Lutein Cell from the Granulosa Follicle Cell during the Preovulatory Stage and under the Influence of Exogenous Gonadotrophins

The granulosa follicle cell of the Graafian follicle of the rabbit ovary differentiates into a lutein cell involved in steroid synthesis. Cytological events which occur within the granulosa cell of the normally stimulated follicle prior to ovulation have been duplicated by the intrafollicular injection of exogenous gonadotrophin. The luteinization of the granulosa cells involves the accumulation of 250- to 300-A, electron-opaque, spherical granules, dispersed within the cytoplasmic matrix, which have been identified as glycogen with the PAS-staining procedure. Further development of the granulosa cell following ovulation involves an increase in cell size, a decrease in the number of RNP particles, and an accumulation of an abundant system of intracellular membranes (agranular endoplasmic reticulum). Glycogen granules first appear in the granulosa cells as the separate, monoparticulate form. After follicle rupture and the formation of agranular endoplasmic reticulum, glycogen particles are present in a rosette arrangement within membrane-bounded vacuoles. The rosette arrangement of glycogen particles is also found dispersed within the cytoplasmic matrix of the lutein cell during the later stages of the cell life-span. Injection of luteinizing hormone or human chorionic gonadotrophin into a mature follicle also produces a marked accumulation of monoparticulate glycogen in the majority of granulosa cells, within 30 min. Cytoplasmic extensions which contain the glycogen masses are noticeably free of RNP particles.     

The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum of UT-1 cells

We have investigated the role of the smooth endoplasmic reticulum (SER) of UT-1 cells in the biogenesis of the glycoprotein (G) of vesicular stomatitis virus (VSV). Using immunofluorescence microscopy, we observed the wild type G protein in the SER of infected cells. When these cells were infected with the mutant VSV strain ts045, the G protein was unable to reach the Golgi apparatus at 40 degrees C, but was able to exit the rough endoplasmic reticulum (RER) and accumulate in the SER. Ribophorin II, a RER marker, remained excluded from the SER during the viral infection, ruling out the possibility that the infection had destroyed the separate identities of these two organelles. Thus, the mechanism that results in the retention of this mutant glycoprotein in the ER at 39.9 degrees C does not limit its lateral mobility within the ER system. We have also localized GRP78/BiP to the SER of UT-1 cells indicating that other mutant proteins may also have access to this organelle. Upon incubation at 32 degrees C, the mutant G protein was able to leave the SER and move to the Golgi apparatus. To measure how rapidly this transfer occurs, we assayed the conversion of the G protein's N-linked oligosaccharides from endoglycosidase H-sensitive to endoglycosidase H-resistant forms. After a 5-min lag, transport of the G protein followed first order kinetics (t1/2 = 15 min). In contrast, no lag was seen in the transport of G protein that had accumulated in the RER of control UT-1 cells lacking extensive SER. In these cells, the transport of G protein also exhibited first order kinetics (t1/2 = 17 min). Possible implications of this lag are discussed.     

Development of chick embryo liver during organ culture: requirement for zinc-insulin

Livers from five-day chick embryos maintained as organ cultures on Eagle's minimum essential medium (MEM) develop an ultrastructure similar to more mature liver cells, except for glycogen deposits and the smooth endoplasmic reticulum (SER) normally associated with such deposits. The enzymes, glycogen synthetase and glycogen phosphorylase, failed to develop in these cells. The addition of zinc-free insulin (insulin-HCl) to MEM promoted the development of small amounts of SER in the cultured cells, as well as an increase in both glycogen synthetase and phosphorylase activities. The addition of zinc-insulin also stimulated an increase in the activities of both enzymes, and promoted the development of greater amounts of SER and the deposition of glycogen, as well. In addition, both forms of insulin not only prevented the fall of total tissue protein which occurs during organ culture on MEM, but also stimulated net protein synthesis in the explanted liver (Benzo and de la Haba, '71).     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver

Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.