Metabolism In Primary Cultures Research Articles

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats.

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.

Effect of ketoconazole and miconazole on 25-hydroxyvitamin D 3 metabolism by cultured chick kidney cells

The antifungal imidazoles, ketoconazole and miconazole, were tested for effects on 25-hydroxyvitamin D 3 metabolism in primary cultures of chick kidney cells. Both behave as competitive inhibitors of 1-hydroxylation of 25-OH-D 3 with approximate K i 's of 0.8 and 5.0 μM for ketoconazole and miconazole, respectively. Ketoconazole was as effective when added at the same time as the substrate as when the cells were preincubated with the compound. Ketoconazole also inhibited the production of 24,25(OH) 2D 3 in cells in which this activity was induced by 1,24(OH) 2D 3. The data suggest that therapeutic doses of these antifungal imidazoles could affect vitamin D status and calcium metabolism.

Thyroid hormone metabolism in primary cultures of fetal rat brain cells

The metabolism of thyroxine 3,5,3′,5′-tetraiodothyronine, (T 4) and 3,5,3′-triiodothyronine (T 3) was studied in primary cultures of dispersed fetal rat brain cells. Cultured brain cells actively metabolized both T 4 and T 3 by enzyme catalyzed deiodination reactions which increase (type II 5′-deiodinase) or decrease (type I 5′-deiodinase and type III 5-deiodinase) the bioactivity of thyroid hormone. Homogenates of cultured brain cells showed both type I and type II 5′-deiodinating activities and these two enzymes tended to differ in their time course of appearance. Cultures exposed to 10 μM cytosine arabinoside for 16 h showed up to a 70% reduction in type I activity without decreasing the type II enzyme suggesting that the type II enzyme is associated with non-dividing neuronal cells. The pre-dominant pathway for T 4 and T 3 metabolism in situ was tyrosyl-ring or type III 5′-deiodination which followed first order kinetics with a t 1/2 of 70 min. T 4 to T 3 conversion by the type II enzyme was consistently observed after correcting for the degradation of newly formed T 3 by the type III enzyme. In situ, both type II and type III enzymes were thiol-dependent and both activities were inhibited by iopanoic acid. Type III 5-deiodination of T 4 produced 34 fmol 3,3,5′-triiodothyronine (rT 3)/h per 10 6 cells at 10 mM dithiothreitol (DTT) and 97 fmol of rT 3/h per 10 6 cells at 50 mM DTT. T 3 production by the II enzyme was 1.2 and 4.4 fmol of T 3/h per 10 6 cells at 10 and 50 mM DTT, respectively. Thyroid hormone deficient culture conditions increased type II enzyme activity by 4–5-fold within 48 h and this was prevented in a dose-dependent fashion by supplementing the media with increasing amounts of T 3. These data indicate that primary cultures of dispersed brain cells mimic the intact cerebral cortex with respect to the metabolism of thyroid hormone and the regulatory mechanisms which defend cerebrocortical T 3 levels. The vigorous metabolism of both T 4 and T 3 by these cultures may explain some of the difficulties in demonstrating thyroid hormone-dependent biochemical changes at physiologically relevant levels of thyroid hormone.

Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH) 2 D 3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with ( 14C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of ( 3H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF 1α, TXB 2, PGF 2α, PGE 2, PGD 2 and PGB 2, the latter being the most abundant one. Indomethacin (10 −5 M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH) 2D 3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B 2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH) 2D 3.

Evidence that levels of malate dehydrogenase and fumarase are increased by cAMP in rat myotubes.

We have investigated the potential role of adenosine 3',5'-cyclic monophosphate (cAMP) in controlling levels of enzymes of energy metabolism in primary cultures of rat skeletal muscle cells. Incubating myotubes with cholera toxin or forskolin (2 persistent activators of adenylate cyclase) significantly increased the levels of two enzymes of oxidative metabolism, fumarase and malate dehydrogenase. These enzymes were also increased (1.5- to 2.0-fold) by phosphodiesterase inhibitors (caffeine, theophylline, theobromine, 3-isobutyl-1-methylxanthine, papaverine, MJ 1988, Ro 20-1724, or SQ 20009) and the cAMP derivatives: 8-bromo-cAMP or dibutyryl cAMP. In contrast two enzymes of glycolytic metabolism, lactate dehydrogenase and pyruvate kinase, were not consistently affected by these agents. The results presented provide strong evidence that an increase in cAMP can lead to an increase in certain enzymes of oxidative energy metabolism.

Steroidogenesis despite a variant metabolism in primary cultures of pig granulosa cells

Enzymic changes in primary cultures of granulosa cells over 9 days were measured and compared with changes occurring during follicular development in vivo. Characteristic of in vivo development of granulosa cells was a large increase in activities of the NADP +-dependent isocitrate, glucose-6-phosphate dehydrogenases and malic enzyme, and smaller increases in the activities of the NAD +-dependent lactate and malate dehydrogenases. In vitro, the NAD +-dependent dehydrogenases increased in activity, while the NADP +-dependent enzymes showed transient or no changes. Despite the uncharacteristic metabolism, granulosa cells in culture could synthesize steroids. Our results suggest that the cells in vitro and in vivo use different metabolic pathways to support syntheses dependent on reducing equivalents.

Differential regulation of copper and zinc metabolism in rat liver parenchymal cells in primary cultures.

A variety of factors influence copper and zinc metabolism in primary cultures of rat hepatocytes. Pronounced elevations in accumulated 64Cu were produced by incubating cells with either Cu2+ or epinephrine with moderate increases produced by incubation with cycloheximide, actinomycin D, or dexamethasone. In addition, copper accumulation was enhanced when cells received culture medium more highly enriched in amino acids and other nutrients. Hepatocytes accumulate greater quantities of 65Zn when incubated with dexamethasone or high extracellular zinc. Higher levels of metallothionein were found in cells incubated with either dexamethasone, zinc, or copper but not with epinephrine. However, epinephrine was effective in elevating ceruloplasmin levels in the cell cytosol. Incubation with dexamethasone or copper-containing medium resulted in elevated 3H-ceruloplasmin in both cytosol and medium. Data presented suggest hormones influence copper and zinc metabolism in liver parenchymal cells in a differential fashion.

Alpha-Adrenergic regulation of enzymes of amino acid metabolism in primary cultures of adult rat hepatocytes.

In primary cultures of rat hepatocytes, addition of dexamethasone (10 microM) plus glucagon (0.5 microM) caused several-fold increases in the activities of serine dehydratase (EC 4.2.1.13), tryptophan oxygenase (EC 1.13.11.11), and tyrosine aminotransferase (EC 2.6.1.5) in 24 h. These inductions were inhibited by insulin. Addition of epinephrine or phenylephrine at 10 microM blocked these inductions. This suppressive effect of adrenergic compounds was completely abolished by the alpha-adrenergic antagonist phenoxybenzamine at 10 microM. Immunochemical analysis with antiserum to serine dehydratase showed that the changes in enzyme activity were due to changes in the amount of enzyme. Epinephrine was effective even when glucagon was replaced by dibutyryl cAMP (50 microM), indicating that alpha-adrenergic suppression of enzyme inductions was mediated by a cAMP-independent mechanism. Furthermore, the findings that prazosin antagonized this epinephrine effect, but yohimbine did not, indicate that the alpha 1- but not the alpha 2-receptor is involved in this inhibition. However, the alpha-adrenergic effect was different from that of insulin in that, unlike the latter, the inductions of tryptophan oxygenase and tyrosine amino-transferase by dexamethasone alone were not inhibited. The alpha-adrenergic action apparently counteracts the action of glucagon and cAMP. For determination of the beta-adrenergic effect of catecholamines on the inductions of enzymes, beta-adrenergic compounds were tested without glucagon. Isoproterenol or epinephrine plus phenoxybenzamine induced tryptophan oxygenase and tyrosine aminotransferase. Induction of serine dehydratase was shown by isoproterenol only in the presence of 1-methyl-3-isobutylxanthine, an inhibitor of phosphodiesterase. These results indicate that catecholamines play dual roles in regulation of the amount of enzyme through their alpha 1- and beta-adrenergic actions.

Regulation of prostaglandin biosynthesis in cultured cells.

Regulation of prostaglandin biosynthesis in cultured cells.

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E 2 and F 2 α did not influence zinc accumulation. Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since 45Ca, 14C-labelled amino acids and [ 35S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [ 35S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.

RNA metabolism in primary cultures of Xenopus laevis kidney cells : III. Processing of rRNA precursor in growing and resting cells

The rate of processing of ribosomal RNA (rRNA) precursor was measured in primary cultures of Xenopus laevis kidney cells in growing and resting states. The kinetic studies on labeling of the precursor RNA and 18 and 28S rRNA in cumulative and pulse-chase experiments revealed that the precursor accumulated and reached a steady state much more rapidly and 18 and 28S rRNA started to appear more rapidly in actively growing cells. Furthermore, the halflives of the precursor RNA were estimated to be 10 and 30 min for growing and resting cells, respectively. These results clearly support the view that not only the transcription and breakdown which have already been shown to be important steps for regulation, but the processing of the precursor RNA molecule would also be controlled upon alteration of the cellular growth rate.

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons: I. Effects of conditioned medium

Neurotransmitter metabolism in primary cultures of dissociated sympathetic neurons from the newborn rat has been examined. Previous studies have shown that neurons grown in the virtual absence of nonneuronal cells developed the ability to synthesize and accumulate radioactive norepinephrine (NE) from [ 3H]tyrosine, but synthesized little [ 3H]acetylcholine (ACh) from labeled choline. However, in the presence of certain types of nonneuronal cells, or in medium conditioned (CM) by them, the neurons produced considerable ACh from choline. Here we show that ACh production from choline depends on the concentration of CM in the growth medium. At 62% CM, the highest level of CM studied, ACh production is >40-fold higher than the control value. Furthermore, homogenates of mixed neuronal/nonneuronal cultures or CM-treated neuronal cultures contain 100- to 1000-fold more choline acetyltransferase activity than neuron-alone sister cultures. On the other hand, the ability to synthesize and accumulate NE is inversely related to the percentage CM in the growth medium; at high concentrations of CM, catecholamine production is depressed about 25-fold from the control. The effects of CM are rather specific, since CM does not affect the number of neurons surviving or their overall growth. These results suggest that CM acts by altering the differentiated fate of individual sympathetic neurons rather than by causing the selective survival or growth of a second population of neurons. CM from primary cultures of several tissues taken from the newborn rat induces neuronal ACh synthesis, as does CM from cultures of several, but not all types of rat cell lines. In contrast, CM from several non-rat cell lines as well as from primary cultures of mouse and chick heart is less effective in causing neuronal ACh production, suggesting some degree of species specificity in this phenomenon.

RNA Metabolism in Primary Cultures of <I>Xenopus laevis</I> Kidney Cells. II. Comparison of the Pattern of RNA Synthesis at Different Phases of Growth

The incorporation pattern of 3H-uridine into various RNA species was examined at the lag, logarithmic and stationary phases in primary cultures of Xenopus laevis kidney cells. The relative amount of labeling of 40S ribosomal RNA precursor was strikingly high in stationary phase cells. It was suggested that in addition to the transcription rate of ribosomal RNA genes, the processing rate of the ribosomal RNA precursor may be reduced when cellular proliferation becomes inactive at the stationary phase.

RNA Metabolism in Primary Cultures of <I>Xenopus laevis</I> Kidney Cells. I. Characterization of Growth Phases and Changes in Number and Size of Nucleoli

Cells from Xenopus kidneys were cultured. Incorporations of 3H-uridine, 3H-thymidine and 14C-protein hydrolyzate were examined, and synthesized RNA was electrophoretically analyzed. The synthesis of RNA, DNA and protein was found to take place at different rates at different phases and the synthesis of ribosomal RNA, as well as other macromolecules, was quite active during the early logarithmic phase. The number and size of nucleoli varied markedly during culture, and these changes were correlated with the activity of cellular RNA synthesis.