Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Changes in Glycoprotein Metabolism in the Cerebral Cortex Following First Exposure of Dark-reared Rats to Light

Abstract: The level of incorporation of [³H]fucose or [³H]lysine into subcel-lular fractions of the visual and motor cortices of 50-day-old dark-reared (D) and light-exposed (L) rats was determined. No differences were found between D and L rats in the incorporation of either precursor into subcellular fractions of the motor cortex, or in any fraction of the visual cortex except the synaptic-membrane fraction. After a 3-h light exposure the incorporation of [³H]fucose into the visual cortex synaptic-membrane fraction was elevated (L/D = 136%). Incorporation of [³H]lysine was elevated in the visual cortex synaptic-membrane fraction of L compared to D rats after a 1-h exposure (L/D = 118%). However, after a 3-h exposure the incorporation was depressed in this fraction (L/D = 79%). No differences could be found in the levels of activity of fucosyl transferase following first exposure to light but dark-rearing itself resulted in increased enzyme activity in the motor cortex compared to normal controls. First exposure of 20-day-old dark-reared rats to light led to an increase in the incorporation of [³H]fucose into soluble glycoproteins of both the visual and motor cortex and into particulate glycoproteins of the visual cortex only. These results are in contrast with those found with 50-day-old animals and suggest that the effects of light-exposure on [³H]fucose incorporation may be age-dependent.     

Patterns of cell differentiation revealed by L-[3H]fucose incorporation in Dictyostelium

A study of the incorporation of l-[6-³H]fucose and d-[6-³H]glucosamine hydrochloride was conducted during the development of the cellular slime mold Dictyostelium discoideum 1-H. Autoradiographs revealed that pulse-labeled vegetative amoebae incorporated [³H]fucose intracytoplasmically within 15 min. The majority of the cells had randomly scattered silver grains but the remainder were distinguished by a dense localized labeling which suggested that oligo or polysaccharide synthesis was occurring. The localized pattern of labeling attributed to active synthesis declines at aggregation and early conus formation. As the pseudoplasmodium makes the developmental transition from the conus to the culmination stages the localized pattern of [³H]fucose labeling was restricted to the prespore cells while the prestalk cells were devoid of label. Prespore vacuoles were not present at the onset of this transition and consequently [³H]fucose incorporation occurred in the cells prior to their differentiation into prespore cells. In contrast to cells composing earlier stages, mature spores exhibited [³H]fucose-containing substances at the cell surface. At appropriate stages certain cells actively synthesize slime and stalk sheath which were labeled with either [³H]fucose or [³H]glucosamine.Prestalk isolates were obtained by transecting migrating slugs. [³H]Fucose was incorporated within 10 min among the basal cells of the isolate in the localized pattern typically found in prespore cells. The incorporation of [³H]fucose occurred prior to prespore differentiation as certain preparations were devoid of prespore vacuoles. Prespore isolates differentiate prestalk cells which have lost the capacity to incorporate [³H]fucose. This investigation suggests that cell contacts and interactions may affect the incorporation of [³H]fucose.     

Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue

1. Surviving sheep colonic mucosal tissue incorporated l-[U-(14)C]threonine when incubated in Krebs medium III at 37 degrees in an atmosphere of oxygen, into a well-characterized mucoprotein fraction, isolated by papain digestion of the incubated scrapings. 2. Acidic hydrolysis and chromatography of the labelled mucoprotein showed that threonine was the only constituent to become labelled. In the presence of puromycin the incorporation of l-[U-(14)C]threonine was considerably diminished (6.7 and 18.5% of control in duplicate experiments). Furthermore, puromycin also decreased incorporation of radioactivity from d-[U-(14)C]-glucose (48.0 and 31.6% of control) and (35)SO(4) (2-) (21.2 and 23.6% of control) into the mucoprotein fraction. 3. In a puromycin-inhibited system, with d-[U-(14)C]-glucose, where the overall specific radioactivity of the mucoprotein was 48% of control, the labelling of the individual monosaccharide constituents (as% of control) was: N-acetylneuraminic acid, 44%; N-glycollylneuraminic acid, 61%; hexosamines, 46%; fucose, 68%; galactose, 34%.     

The biosynthesis of intestinal mucins. The effect of salicylate on glycoprotein biosynthesis by sheep colonic and human gastric mucosal tissues in vitro

1. Incubation of sheep colonic mucosal scrapings in Krebs-Ringer buffer for 2(1/2)hr. in the presence of salicylate (15mm) resulted in decreased incorporation of radioactivity into the epithelial glycoprotein from the following labelled precursors: 16.6mum-d-[2-(14)C]glucose (83.9% inhibition), 20mum-l-[U-(14)C]threonine (82%) and (35)SO(4) (2-)(79%). Oxygen uptake measured simultaneously was diminished to 41% of the control value. 2. At lower concentrations of salicylate (e.g. 3.75mm), incorporation of 20mum-l-[U-(14)C]threonine was little affected (3-6% inhibition), whereas utilization of 4mum-d-[U-(14)C]glucose and (35)SO(4) (2-) was inhibited (41-48% and 40-59% of the control values respectively). 3. Analysis of the papain-digested glycoprotein from tissue incubations with 16.6mum-d-[2-(14)C]glucose in the presence of salicylate (3.75mm) showed large decreases in labelling of N-acetylneuraminic acid and N-glycollylneuraminic acid residues (57% and 34% of the control values respectively) and of hexosamine constituents (glucosamine, 55% inhibition; galactosamine, 33% inhibition). Labelling of neutral sugars (galactose and fucose) was relatively little affected (9 and 11% inhibition respectively). 4. Glucose 6-phosphate transaminase and glucosamine 6-phosphate acetylase in particle-free enzyme preparations of the sheep tissue were unaffected by salicylate at the above concentrations. Acetyl-CoA synthetase was markedly inhibited. 5. Human gastric mucosa (from operation), on incubation as above, had in one experiment an oxygen consumption of 9.9mul./hr./mg. dry wt. of tissue and incorporated 5mum-d-[U-(14)C]glucose (15.8% of the total radioactivity added) into bound hexosamine (20.6% of the total radioactivity incorporated), hexoses (glucose and galactose, 5.7%) and fucose (14.2%). The presence of salicylate (15mm) decreased the incorporation of 5mum-d-[U-(14)C]glucose into the glycoprotein by 74%, all sugar constituents being affected, without influence on the rate of oxygen consumption. 6. The results suggest an inhibitory effect of salicylate on glycoprotein biosynthesis at the level of the amino sugar intermediates.     

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.     

Rapidly metabolized glycoproteins in a neuroplastoma cell line

The metabolism of neuroblastoma cell glycoproteins was examined using l-[³H]fucose. Incubation of monolayer cultures with [³H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [³H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h)_ of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [³H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 · 10^?4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [³H]fucose. Although the major proportion of the radioactivity remained as [³H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components. However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [⁴C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

Biosynthesis of piperlongumine

The incorporation of l-[U-¹⁴C]lysine and l-[U-¹⁴C]phenylalanine into piperlongumine has been demonstrated in Piper longum. The subsequent stepwise degradation to methyl-(3,4,5-trimethoxyphenyl)-propanoate and δ-aminovaleric acid revealed that the C₆-C₃ moiety of the alkamide arises from phenylalanine; the heterocyclic ring is biosynthesised from lysine. It has also been shown that dl-[2-¹⁴C]tyrosine and [2-¹⁴C]sodium acetate are poor precursors of piperlongumine.     

RAPID TRANSPORT OF FUCOSYL GLYCOPROTEINS TO NERVE ENDINGS IN MOUSE BRAIN

Abstract— Mice were injected intracerebrally with mixtures of [³H]fucose and [¹⁴C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [³H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [¹⁴C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase.     

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.     

Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.