An autoradiographic study of calcium transport in spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The route of calcium transport to the sites of spicule formation in the gorgonian Leptogorgia virgulata has been examined by the use of ⁴⁵Ca as a tracer in light- and electron-microscopic autoradiography. From 1 to 15 min after the 15-min incubation the tracer accumulates in the axis. After 15 min there is a movement of label out of the axis largely to the peripheral region of the axis, the axial epithelium, and the mesoglea. By 60 min much of the label is in the spicules reaching its maximum level at 120 min. When calcium enters the scleroblast from the mesoglea, it appears to be transported to the spicule by electron-dense bodies. There does not appear to be a simultaneous release of all ionic calcium from the axis, but rather a continuously increasing efflux which levels off at 60–120 min. Not all of the calcium reaching the axis will traverse it en route to spicules; instead, a portion of it apparently precipitates as an amorphous compound.Contribution No. 550; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208 USA     

Ultrastructural investigation of spicule formation in the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Ultrastructural examination of original and regenerated branch tips of the gorgonianLeptogorgia virgulata reveals that spicule formation begins with the aggregation of scleroblasts in the mesoglea. Calcite crystal deposition occurs within a Golgi vacuole containing organic matrix. Vacuole size increases while matrix incorporation and subsequent crystal growth continue, filling the vacuole. At approximately this time, the scleroblasts dissociate and wart formation begins. Further spicule growth stretches the cell into a thin envelope. Fusion of vacuole and plasma membrane followed by breach formation during spicule growth, as well as scleroblast atrophy or migration from mature spicules, result in the transition of the spicule from the intracellular to the extracellular environment. The results also reveal aborted spicules and digestive bodies, implying possible relationships among calcification, detoxification, and waste management.Contribution No 436, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla

Summary The synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-³H and L-3,4-dihydroxy [ring 2,5,6-³H] phenylalanine (L-dopa³H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine ³H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine ³H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa ³H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine ³H or L-dopa ³H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals. Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group of Hypertension of the Clinical Research Institute of Montreal and by the Canadian Heart Foundation     

Ultrastructural cytochemistry of atrial muscle cells

Summary Sections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 5 min, most of the label had migrated from the RER to the Golgi complex. Some label was already present over specific granules by 2 min but the peak was reached at 1 h. By 4 h, the label over the specific granules had diminished, possibly indicating a release of newly-synthetized secretory material outside the cell. The label over myofilaments and Z-bands was relatively high at most time intervals, suggesting an early and important incorporation of leucine into the contractile and structural proteins of these organelles. The label over the cytosol was initially high and increased even further at 5 and 20 min but decreased to a very low level at 4 h. In contrast, the label over the cell surface rose continuously and peaked at 4 h. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the cytosol before reaching their destination.     

Ultrastructural radioautography of the incorporation of tritiated leucine by the rat adrenal medulla in vivo

Summary Sections of tissues from the adrenal medullae of young rats were subjected to radioautography after a single intravenous injection of L-leucine 4,5 ³H to identify the sites of synthesis and follow the migration of newly-formed proteins in both adrenaline-storing (A) and noradrenaline-storing (N) cells. As early as 2 min after injection of leucine ³H, the label was highest in the rough endoplasmic reticulum (RER) of A and N cells, suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the RER into the Golgi complex of both cell types. Some label was already present over the secretory granule matrix (chromogranins) by 2 min but the peak was reached at 1 h in both A and N cells. By 4 h, the label over the secretory granules had diminished, indicating a release of newly-synthetized chromogranins outside the cells. The label over the hyaloplasm was relatively high at 2 min but it decreased rapidly to low levels. In contrast, the label over the cell surface continually increased to reach the highest levels among all organelles at 4 h in both cell types. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the hyaloplasm, before reaching the surface of A and N cells.Supported in part by the Quebec Heart Foundation, the Medical Research Council of Canada (Grant MT-1973), the J.-L. Levesque Foundation, the Ministry of Education of Quebec (Formation de Chercheurs et Action Concertée) and the Fond de l'Université de Montréal (Cafir)     

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats

Summary Sections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine3,5 ³H or of L-fucose ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4h. After injection of L-fucose ³H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose ³H and tyrosine ³H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.Recipient of a summer fellowship from the Kidney Foundation of Canada     

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta)

Summary Frog pancreatic tissue was pulse-labelled in vitro with ³H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions.Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae.Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues.The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30° C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.     

Glycoprotein transport in the surface mucous cells of the rat stomach

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.     

MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF B CELLS OF FETAL RAT ENDOCRINE PANCREAS IN ORGAN CULTURE : Evidence for (Pro)Insulin Biosynthesis

Fetal rat pancreases explanted on the 18th day of gestation and maintained in organ culture for 1–10 days were utilized for this series of studies. Ultrastructurally, at the time of explantation, the majority of fetal B cells was sparsely granulated and characterized by numerous free ribosomes and undeveloped rough endoplasmic reticulum (RER) and Golgi complexes. During the culture period, extensive development of the RER and Golgi complexes preceded an increasing accumulation of β-granules. This later increase in the number of β-granules and in the concentration of immunoreactive insulin was paralleled by a reduction of RER and Golgi complex activity. High resolution radioautographic studies of pulse-chase experiment over a 1 hr period demonstrated the shift of silver grains from the elements of the RER, through the Golgi region, and finally to the β-granules. Incubation with ¹⁴C-labeled leucine demonstrated the incorporation of radioactivity into molecules possessing the immunological and electrophoretic properties of insulin. These studies indicate that de novo synthesis of (pro)insulin occurs also during culture of fetal rat pancreas explanted relatively late in gestation.     

Intracellular transport of proteins in active and resting secretory cells of the venom gland of Vipera palaestinae

The intracellular transport of venom proteins has been studied in active and resting venom glands of the snake Vipera palaestinae by electron microscope radioautography after an intra-arterial injection of [3H]leucine. In the active gland, most of the label is initially (10 min) found over the RER. By 30 min, the relative grain density of the Golgi complex reaches its maximum, with concomitant increase in the labeling of the condensing vacuoles. Later on, a steep increase in radioactivity of the secretory granules is observed. At 3 h, these granules, which comprise about 2% of the cell volume, contain 22% of the total grains. At the following hour, their labeling declines and at the same time the radioactivity of the secreted venom is increased. It is concluded that, in the active cell, venom proteins are transported via the Golgi apparatus into membrane-bounded granules which are the immediate source of the secreted venom. An alternative pathway, which involves the RER cisternae as a storage compartment, seems unlikely, since incorporated label does not accumulate in this compartment after prolonged postpulse intervals. The route of intracellular transport of proteins in the resting glands is similar to that of the active ones, but the rate of synthesis and transport is much slower. The present results and earlier data, thus, show that the increase in the rate of secretion after initiation of a new venom regeneration cycle is the result of accelerated rates of both synthesis and transport.     

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.     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.     

Seasonal localization of a collagenous protein in the organic matrix of spicules from the gorgonian Leptogorgia virgulata (Cnidaria: Gorgonacea)

Spicules of the gorgonian Leptogorgia virgulata possess an insoluble matrix fraction that is predominantly collagenous in summer months. This collagenous component is largely absent in winter months. Using an antibody directed against the 140 kD collagenous protein (CP) of the insoluble matrix, immuno-gold labelling was employed to localized this protein at the transmission electron-microscopy level throughout the year, and in different areas of the gorgonian colonies. Within the tip regions, the 140 kD CP varied throughout the year in the spicules, electron-dense bodies (EDBs) of scleroblasts, polyp vesicles, desmocytes and axes. In the mid and base regions, the 140 kD CP varied throughout the year in the spicules, EDBs and lysosomes of scleroblasts, desmocytes and axes. This variation in the location and density of the label suggests a dynamic annual cycling of the collagenous component of the insoluble matrix. EDBs may transport a collagenous component of the matrix to the spicule-forming vacuole. A component of the 140 kD CP may be transported and/or degraded by polyp vesicles and lysosomes, respectively. The pattern of labelling of the axial region suggests that translocation and storage of a component of the collagenous protein may occur. Environmental factors may be responsible for the triggering of matrix cycling.     

PROTEIN SYNTHESIS,STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL : An Autoradiographic Study

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H³ as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H³; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H³ can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.     

Morphological effects of brefeldin A on the intracellular transport of secretory materials in parotid acinar cells

The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPPase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organelles were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.     

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.     

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.     

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).