THE ELABORATION OF PROTEIN AND CARBOHYDRATE BY RAT PARATHYROID CELLS AS REVEALED BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The parathyroid glands of young rats were radioautographed after a single injection of the protein precursor tyrosine-³H in the hope of identifying the sites of synthesis and migration of newly formed protein in the gland cells. The same procedure was used after injection of the glycoprotein precursor galactose-³H. As early as 2 min after intravenous injection of tyrosine-³H, the label was mainly found in the rough endoplasmic reticulum suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the rough endoplasmic reticulum into the Golgi apparatus. By 20 and 30 min, some label had migrated from there into secretory granules. By 45 min and 1 hr, the label content of the cell had decreased, indicating release of labeled material outside the cell. At 2 min after intravenous injection of galactose-³H, the label was mainly present in the Golgi apparatus, where presumably galactose is taken up into glycoprotein. By 10 min, some label appeared in secretion granules and by 30 min release of the material to the outside of the cell was under way. In conclusion, it is likely that the tyrosine-labeled protein material consists mainly of the parathyroid hormone. The galactose-labeled carbohydrate material would be either associated with the hormone in the cell or be part of a distinct glycoprotein which may be the one present on the outer surface of the plasma membrane (cell coat).     

LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION : II. Electron Microscopic Radioautographic Study of the Mouse Lactating Mammary Gland

In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-³H or 9, 10-palmitic acid-³H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

ELABORATION OF THE MATRIX GLYCOPROTEIN OF ENAMEL BY THE SECRETORY AMELOBLASTS OF THE RAT INCISOR AS REVEALED BY RADIOAUTOGRAPHY AFTER GALACTOSE-3H INJECTION

The elaboration of enamel matrix glycoprotein was investigated in secretory ameloblasts of incisor teeth in 30–40-g rats. To this end, the distribution of glycoprotein was examined histochemically by the use of phosphotungstic acid at low pH, while the formation of glycoprotein was traced radioautographically in animals sacrificed 2.5–30 min after galactose-³H injection. Histochemically, the presence of glycoprotein is observed in ameloblasts as well as in the enamel matrix; in ameloblasts glycoprotein occurs within the Golgi apparatus in amounts increasing from the outer to the inner face of the stacks of saccules, and is concentrated in condensing vacuoles and secretory granules; in the enamel matrix, glycoprotein is observed within linear subunits. Radioautographs at 2.5 min after injection demonstrate the uptake of galactose-³H label by Golgi saccules, indicating that galactose-³H is incorporated into glycoprotein within this organelle. After 5–10 min, the label collects in the condensing vacuoles and secretory granules of the Golgi region. By 20–30 min, the label appears in the secretory granules of the apical (Tomes') processes, as well as in the enamel matrix (next to the distal end of the apical processes, and at the tips of matrix prongs). In conclusion, galactose contributes to the formation of glycoprotein within the Golgi apparatus. The innermost saccules then distribute the completed glycoprotein to condensing vacuoles, which later evolve into secretory granules. These granules rapidly migrate to the apical processes, where they discharge their glycoprotein content to the developing enamel.     

EVIDENCE FOR THE PARTICIPATION OF THE GOLGI APPARATUS IN THE INTRACELLULAR TRANSPORT OF NASCENT ALBUMIN IN THE LIVER CELL

A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-¹⁴C or leucine-³H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-¹⁴C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-¹⁴C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at ~20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golgi fraction than in the microsomes. By radioautography the silver grains were preferentially localized over the rough-surfaced endoplasmic reticulum at the 5 min interval. Apparent activity in the Golgi zone was noted 9 min after the injection; at 15 and 20 min, the majority of the grains were found in this location. Many of the grains associated with the Golgi apparatus were located over Golgi vacuoles containing 300–800 A electron-opaque bodies. It is concluded that albumin is synthesized on bound ribosomes, subsequently is transferred to the cavities of rough-surfaced endoplasmic reticulum, and then undergoes migration to the smooth-surfaced endoplasmic reticulum and the Golgi apparatus. In the latter organelle, albumin can be expected to be segregated together with very low density lipoprotein in vacuoles known to move toward the sinusoidal portion of the cell and release their content to the blood.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.     

INTRACELLULAR ACCUMULATION OF PROTOCOLLAGEN AND EXTRUSION OF COLLAGEN BY EMBRYONIC CARTILAGE CELLS

The synthesis of collagen can be interrupted, after the assembly of proline-rich and lysine-rich polypeptide chains called protocollagen, by incubating connective tissues anaerobically. Under these conditions the proline and lysine residues in protocollagen are not hydroxylated to hydroxyproline and hydroxylysine, and protocollagen molecules accumulate intracellularly. Chemical data and radioautographs at the level of the light and electron microscopes indicated that in tissues labeled with proline-3,4-³H under nitrogen, there appeared to be an accumulation of radioactivity over the ground cytoplasm. When the inhibition of protocollagen hydroxylase was reversed by exposing the tissue to oxygen, the accumulated protocollagen-³H was converted to collagen-³H and there was a rapid transfer of label from the ground cytoplasm to the extracellular matrix. There was no significant change in distribution of label over either the Golgi vacuoles or the cisternae of the endoplasmic reticulum. The failure to find a significant change in distribution of label over the Golgi vacuoles or the cisternae does not completely exclude the possibility that these two compartments are involved in the extrusion, but the data are consistent with the simpler notion that the completed collagen molecules pass directly from the ground cytoplasm to the extracellular matrix.     

RADIOAUTOGRAPHIC ANALYSIS OF THE SECRETORY PROCESS IN THE PAROTID ACINAR CELL OF THE RABBIT

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-³H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.     

CYTOLOGICAL STUDIES ON TWO FUNCTIONAL HEPATOMAS : Interrelations of Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes

The Reuber hepatoma H-35 and Morris hepatoma 5123 have been studied by electron microscopy and by cytochemical staining methods for a number of phosphatases. These studies emphasize the resemblances of the two tumors to rat liver, but they also indicate distinctive features in each of the three tissues. Secretory product accumulates within the cisternae of the Golgi apparatus that dilate to form the Golgi vacuoles. The vacuoles apparently separate, and secretory material undergoes further condensation within them. These "secretory vacuoles" possess acid phosphatase activity and may thus be considered lysosomes. The membranes of the Golgi apparatus are without acid phosphatase activity but show high levels of thiaminepyrophosphatase activity. The endoplasmic reticulum also hydrolyzes thiaminepyrophosphate but at a lower rate; it hydrolyzes the diphosphates of uridine, guanosine, and inosine rapidly. These observations and the electron microscopic images are consistent with the view that the cytomembranes are in a dynamic state of flux, movement, and transformation in the living cell, and that smooth surfaced derivatives of the endoplasmic reticulum become refashioned into the Golgi membranes as the Golgi membranes are being refashioned into those that delimit secretory vacuoles. The variations encountered in the two hepatomas are described. The electron microscope literature dealing with the relations of the Golgi apparatus to secretory granules, on the one hand, and the endoplasmic reticulum, on the other, is reviewed briefly.     

Sites of lipoprotein lipase activity in adipose tissue perfused with chylomicrons. Electron microscope cytochemical study

Lipoprotein lipase activity was studied in rat parametrial adipose tissue perfused with chylomicrons and in gelatin blocks containing postheparin plasma and chylomicrons. The tissues and blocks were fixed in glutaraldehyde and incubated in 0.035 M CaCl₂-0.1 M Tris medium (pH 8.3) at 38°C. The doubly labeled chylomicron triglycerides (glycerol-³H and palmitate-¹⁴C) in the tissues and blocks were hydrolyzed during incubation to free fatty acids (FFA) and the FFA remained in the specimens; hydrolysis was inhibited by 0.004 M diethyl paranitrophenyl phosphate (E-600). Incubated blocks and tissue were treated with 0.05 M Pb(NO₃)₂, postfixed in OsO₄, dehydrated with acetone, embedded in Epon, and examined by electron microscopy. The incubated blocks contained electronlucent areas and granular and laminar precipitates at sites of hydrolysis. Similar precipitates were found in incubated tissue, within vacuoles and microvesicles of capillary endothelium, and in the subendothelial space (between the endothelium and pericytes), but not in the capillary lumen or in or near fat cells. The cytochemical reaction was greatly reduced, in blocks and tissues incubated with E-600. It is concluded that plasma glycerides are hydrolyzed by lipoprotein lipase in capillary endothelial cells and in the subendothelial space of adipose tissue and that glycerides across the endothelial cells within a membrane-bounded system.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

ON THE SITE OF SULFATION IN COLONIC GOBLET CELLS

The location of bound S³⁵ in the goblet cell of the rat colon at time points from 2 to 60 minutes after administration of S³⁵ as sodium sulfate has been observed in vivo and in vitro by radioautographic techniques. Grains were first observed by electron microscopy over the stacked lamellae of the paranuclear part of the Golgi apparatus. The label was subsequently found associated with the supranuclear Golgi lamellae and was then seen associated with the smooth membranes limiting the mucin granules in the goblet. Finally, between ½ and 1 hour, the secreted mucus product in the crypts became radioactive. Neither mitochondria nor the endoplasmic reticulum was labeled. It is concluded that the Golgi apparatus is the organelle in which sulfation occurs.     

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well- developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post- Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

A RADIOAUTOGRAPHIC STUDY OF GLYCERIDE SYNTHESIS IN VIVO DURING INTESTINAL ABSORPTION OF FATS AND LABELED GLUCOSE

Radioautography was used to detect the synthesis of labeled glycerides in intestinal absorptive cells following injections of fatty chyme and glucose-6-H³ into ligated segments of upper jejunum of fasting rats. Absorption intervals ranged from 2 to 20 min. Labeling is evident throughout the cells in as short a time as 2 min. Most grains are present over droplets of absorbed fat beginning with those in the endoplasmic reticulum immediately subjacent to the terminal web. With longer absorption periods, frequent grains are present over accumulations of fat droplets in the Golgi cisternae and intercellular spaces. A similar pattern of grains is seen following absorption of either linoleic acid or safflower oil. By comparison, considerably less label is present in the cells when the fat is extracted with alcohol prior to radioautographic procedures, or when labeled glucose alone is absorbed. A significant incorporation of glucose label into newly synthesized glycerides is indicated and confirmed by scintillation counts on saponified lipid extracts. The grain distribution implies an involvement of the extreme apical endoplasmic reticulum in this synthesis.     

Affinity cytochemical differentiation of glycoconjugates of small intestinal absorptive cells using Pisum sativum and Lens culinaris lectins

We studied the subcellular localization of glycoconjugates recognized by the garden pea and lentil lectins (Pisum sativum, PSA; Lens culinaris, LCA) in mature absorptive cells of duodenum and jejunum of fasted rats. PSA and LCA are mannose-, glucose-, and N-acetyl-glucosamine-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. The binding reactions were cytochemically demonstrated in a pre-embedment incubation system using peroxidase-labeled lectins. Both pea and lentil lectins bound with constituents of nuclear envelope and endoplasmic reticulum, cisternae of the Golgi apparatus, several Golgi-associated vesicles, lysosomes, and portions of the plasma membrane. PSA and LCA label was non-homogeneous in the endoplasmic reticulum; in the Golgi apparatus the reactions were most intense in the cis and medial cisternae of the stacks. For inhibition of the intense reactions apparent in the Golgi apparatus, in lysosomes, and at the plasma membrane, considerably higher concentrations of competitive sugars were necessary than for abolition of the endoplasmic reticulum label. This indicates that endoplasmic reticulum glycoconjugates bind at low affinities with pea and lentil lectins, and that high-affinity PSA/LCA-binding glycoconjugates, which may correspond to corefucosylated N-linked glycans, predominate in cis and medial Golgi cisternae, lysosomes, and at the plasma membrane.