Ascorbic acid accumulation by cultured rat adrenocortical cells

The influence of adrenocorticotrophin (ACTH) on radiolabeled ascorbic acid (AA) accumulation by adrenocortical cells was examined in primary cultures of collagenase dissociated glands from adult male rats. The cells were ACTH responsive by morphological and steroidogenic criteria. After 5 d in AA-free medium, cells pretreated with 100 mU/ml ACTH for 3 d took up two to three times more AA over a 2 h period than did untreated controls (4.0 to 10.0 nmol versus 1.7 to 3.4 nmol AA/micrograms DNA). In contrast, ACTH administered on Day 6 concurrently with AA inhibited AA accumulation compared to cultures exposed to AA alone. This acute inhibitory effect of ACTH was in the order of 30% in cultures pretreated with ACTH for 3 d but was not significant (7%) without ACTH pretreatment. The results show that ACTH has distinct long term stimulatory and acute inhibitory effects on AA accumulation by adrenocortical cells and suggest that both maximal AA accumulation and the responsiveness to acute inhibition of AA accumulation by ACTH may depend on the maintenance of the differentiated state of the adrenal cortex.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

Characterization of the rat adrenal medulla cultured in vitro

Summary A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.     

Ascorbic acid facilitates chicken myoblast fusion in vitro

Summary Addition of low concentrations of ascorbic acid (5 μg/ml to 10 μg/ml) to myogenic chick embryo cultures resulted in an early fusion. At 30 h cultures receiving small amounts of ascorbic acid presented fusion rates 3 times that of the control. However, control rates of fusion were not different from those of experimentals at 50 h. No such effect was seen with ascorbic acid added at 24 h of culture, or with ascorbic acid addition to a calcium-deprived system. These findings demonstrate that the calcium binding properties of ascorbic acid can induce precocious myogenic fusion.     

Effects of prolonged ACTH-stimulation on adrenocortical accumulation of lipofuscin granules in aged rats

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Ascorbic acid and development of xylem and phloem cells in the pine trunk

Changes in the levels of ascorbic acid (AA), its oxidized form, dehydroascorbic acid (DHA), and uronic acids as initial precursors for the AA synthesis were studied as related to the degree of xylem and phloem cell development in the course of early and late wood formation in the trunks of Scots pine (Pinus sylvestris L.). The cells of mature and conducting phloem, cambial zone, differently developed cells in the zones of cell enlargement and maturation were obtained by successive scraping tissue layers from trunk segments of 20–25-year-old trees; tissue identification was checked anatomically and histochemically. The contents of compounds tested were calculated per dry weight and per cell basis. We found great differences in the contents of AA and DHA and also in their ratio in dependence of the wood type developing in the pine trunks during growth period and on the stage of differentiation of xylem and phloem cells. Changes in the AA content during xylem cell differentiation were accompanied by changes in the content of uronic acids. The amounts of AA, DHA, and uronic acids were the highest at the stage of early lignification and reduced with tracheid maturation. The AA to DHA ratio changed differently in the course of early and late xylem lignification. It reduced from the start of lignification to the formation of early mature xylem and, in contrast, increased in mature late wood; this indicates a difference in the level of redox processes in these tissues.     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

Density separation of rat adrenocortical cells: Morphology,steroidogenesis, and P-450scc expression in primary culture

Summary We have developed a method that separates rat adrenocortical cells by density into populations which retain zone specific properties in primary culture. Two different parenchymal populations were obtained and designated 2FASC (1.034 g/ml, 18.0 μm cell diameter) and 7GLOM (1.069 g/ml, 11.7 μm cell diameter). In freshly isolated cell suspensions the physical characteristics and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that 2FASC cells originated predominantly from the zona fasciculata and 7GLOM cells from the zona glomerulosa. In primary culture (Dulbecco's Modified Eagle's Medium-F12 medium with 15% horse serum and 2.5% fetal bovine serum) the two populations exhibited different morphologies. 2FASC cells retained lipid and formed cohesive epithelial monolayers that remained stationary for 3 wk. 7GLOM cells were initially epithelial but rapidly lost lipid, spread, and assumed fibroblastic shapes. Both cell types were ositive for the cholesterol side-chain cleavage cytochrome P-450 by immunofluorescence. Therefore, the morphologic changes seen in 7GLOM cultures were due to modulation, not fibroblastic overgrowth. This phenotypic plasticity may reflect the mesodermal origin of the adrenal cortex, and the subcapsular location of 7GLOM cells in vivo. In contrast, cells such as 2FASC which are located deeper in the cortex seem to have a more restricted, fully committed parenchymal phenotype. This work was supported by a studentship to C. D. R., and by a grant and research associateship to N. A., from the National Cancer Institute of Canada.     

Neonatal rat heart cells cultured in simulated microgravity

Summary In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA- designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organizations of cardiac cells in vitro.     

Ascorbic acid and beta-carotene reduce stress-induced oxidative organ damage in rats

Antioxidants are potential therapeutic agents for reducing stress-induced organ damage. We investigated the effects of ascorbic acid and β-carotene on oxidative stress-induced cerebral, cerebellar, cardiac and hepatic damage using microscopy and biochemistry. Male Wistar albino rats were divided into five groups: untreated control, stressed, stressed + saline, stressed + ascorbic acid and stressed + β-carotene. The rats in the stressed groups were subjected to starvation, immobilization and cold. The histopathological damage scores for the stressed and stressed + saline groups were higher than those of the control group for all organs examined. The histopathological damage scores and mean tissue malondialdehyde levels for the groups treated with antioxidants were lower than those for the stressed and stressed + saline groups. Mean tissue superoxide dismutase activities for groups that received antioxidants were higher than those for the stressed + saline group for most organs evaluated. Ascorbic acid and β-carotene can reduce stress-induced organ damage by both inhibiting lipid oxidation and supporting the cellular antioxidant defense system.     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

Ascorbic acid induces spectrin reorganization in bull epididymal spermatozoa

The acrosome, a complex organelle, plays a key regulatory role in the sperm–egg interaction. We have previously shown that ascorbic acid affects both motility and spectrin protein patterns in sperm. In this study, we further characterized the changes in spectrin in sperm challenged with ascorbic acid, using SDS-PAGE, western blots, and immunofluorescence. Ascorbic acid shifts spectrin to a higher-molecular-weight species based on western blot studies. This shift in the spectrin band correlates with a striking series of changes in spectrin immunofluorescence patterns. Upon ascorbic acid challenge, spectrin localization changes, eventually resulting in the formation of vesicles. These vesicles can reach sizes up to five times the original volume of the sperm cell and sometimes show multiple spikes. These findings indicate that a novel process is taking place in the acrosome upon ascorbic acid challenge and suggest that the cytoskeleton may be a useful target for studying and hopefully controlling the sperm–egg interaction. This revised version was published online in July 2006 with corrections to the Cover Date.     

乳酸对大鼠大脑皮质神经元存活率的影响

目的:探讨乳酸对原代培养的皮质神经元存活率的影响.方法:体外原代培养大鼠大脑皮质神经元,将神经元分为两部分:①添加不同浓度乳酸(0,5,10,20 mmol/L)使培养液pH值分别降至7.35,7.15,6.95和6.00,观察神经元存活率;②添加不同浓度乳酸(0,5,10,20 mmol/L)后,保持培养液pH值为7.35,分别观察神经元存活率,分析乳酸对神经元的作用是通过下调pH值还是乳酸本身的作用.结果:①神经元存活率随pH降低而降低,与pH值为7.35组相比差异有显著性.②不同乳酸浓度对神经元存活率也有影响,神经元存活率降低,与对照组相比差异明显.③pH下调组神经元存活率显著低于相应乳酸作用组.结论:乳酸升高造成pH降低与神经元存活率关系密切,过量乳酸亦可不依赖下调pH值的作用发挥神经毒性.     

Ascorbic acid in pollen: Conversion of L-galactono-1,4-lactone to L-ascorbic acid by Lilium longiflorum

The L-ascorbic acid (AA) content of pollen from three cultivars of Lilium longiflorum Thunb. was 260–280 μg/g fresh wt. of pollen. During germination ascorbic acid content gradually decreased reaching 70% of the original value at 6 h. Pollen germinated in media containing 0.29 M D-glucose (an osmoregulator and carbon source) failed to synthesize ascorbic acid but pollen germinated in 0.29 M pentaerythritol (a non-metabolizable osmoregulator) supplemented with L-galactono-1,4-lactone (L-GalAL) did form ascorbic acid, dependent upon the concentration of the lactone. Lycorine inhibited germination but had negligible effect on the conversion of L-GalAL to AA.     

Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells

Cordyceps sinensiscontains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis(CS) and thesignaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximun at 25 μg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis. J. Cell. Biochem. 69:483–489, 1998. © 1998 Wiley-Liss, Inc.     

Primary culture of normal rat adrenocortical cells

Summary Rat adrenocortical cells retiained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution ofd-valine in place ofl-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [³H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described. This work was supported by Grant CA-16417 from the National Cancer Institute.     

Ascorbic acid improves conversion of white spruce somatic embryos

Summary The effects of exogenous applications of ascorbic acid on white spruce somatic embryogenesis were examined. Increasing concentrations of ascorbate (1 μM to 100 μM) in the germination medium enhanced somatic embryo conversion in a linear fashion. At the optimal ascorbate level (100 μM) the number of embryos able to undergo normal conversion, i.e., emergence of both root and shoot, increased from 34% (control) to 58%. The effect of ascorbate had a more pronounced effect on shoot growth than on root emergence; and at 100 μM ascorbate, the percentage of embryos able to produce new leaf primordia increased from 47% (control) to 79%. Root emergence increased slightly from 64% in the control embryos to 74% in the presence of ascorbic acid. The ascorbate-treated embryos were characterized by an enlarged apical region, presumably due to a larger number of leaf primordia produced, and by dark green leaves. When allowed to grow further, these embryos were able to develop into normal plantlets.     

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

Summary The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.