Replacement of serum with a defined medium increases β-adrenergic receptor number in cultured glioma cells

β-Adrenergic receptors of C6 glioma cells were compared following growth in serum or in a serum-free defined medium. In the absence of serum there was an increase in β-receptor number by approx. 50% as measured by the binding of [¹²⁵I]iodohydroxybenzylpindolol. Other receptor properties, such as the affinities for β-adrenergic agonists and antagonists, were not different in the two growth conditions. The alteration in receptor number took over 3 days to occur and was not readily reversible. The increase in β-adrenergic receptor number of cells grown in a serum free medium indicates that replacement of serum with a defined medium is able to alter the expression of at least one cell surface marker, the β-adrenergic receptor. These results imply that growth of cells in defined media yields cells with properties of the plasma membrane that differ from those of cells grown in serum-containing media.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

Adrenergic receptor characteristics of cardiac myocytes cultured in serum-free medium: comparison with serum-supplemented medium

Neonatal rat ventricular myocytes cultured in serum-free medium coexpress both alpha 1 and beta 1 receptors as determined by radioligand binding studies. In cells exposed to serum for 48 hr surface area increased 3.69 fold, but the maximum number of binding sites ([125I]-iodocynanopindolol) only increased 1.5 fold from 12956 +/- 7579 to 19676 +/- 5181 sites/cell (n = 5, p less than .05) yielding a value of 2.48 sites/um2 for cells grown in serum-supplemented medium compared with 6.96 sites/um2 for cells grown in serum-free medium. Thus serum-induced hypertrophy is associated with a decrease in beta 1 receptor density relative to cell size; however, adenylate cyclase response is unaffected. This cell culture system constitutes an excellent model for studying interventions that may influence the regulation of cardiac myocyte hypertrophy by nonhemodynamic factors, particularly through the adrenergic receptor system.     

Serum catecholamines desensitize beta-adrenergic receptors of cultured C6 glioma cells

C6 glioma cells grown in medium containing fetal bovine serum have a decreased beta-adrenergic receptor number and beta-receptor-stimulated cyclic AMP accumulation as compared to cells grown in a serum-free, defined medium. The decreased number of receptors and decreased cAMP accumulation are attributable to a suppression of receptor binding and response by serum as opposed to increases produced by growth in the defined medium. Serum, when added to cells grown in the absence of serum, stimulated cellular cyclic AMP levels to 2-3 times basal levels. This direct stimulatory effect was blocked by incubation of the cells with the beta-adrenergic antagonist propranolol and was partially reversed by dialysis of the serum. In contrast, addition of serum to cells that have been grown with serum fails to stimulate cyclic AMP accumulation. The decrease in receptors following growth in serum can be mimicked by growing cells in serum-free medium in the presence of beta-adrenergic agonists such as isoproterenol or norepinephrine. Radioenzymatic assays indicate that fetal bovine serum contains approximately 0.3 nM norepinephrine and lower concentrations of epinephrine. It thus appears that growth of C6 cells in serum-containing media desensitizes the beta-adrenergic receptor/cyclic AMP system of these cells. This desensitized state appears to result primarily from the action of catecholamines present in serum. These data indicate that retained catecholamines are one component in serum that can modify expression of beta-adrenergic receptors and hormonal response of cultured glioma cells.     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

1,4-Dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts

By using the radioactively labeled 1,4-dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non-interacting DHP binding sites (Bmax, 1.2 +/- 0.3 pmol/10(6) cells; Kd, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half-time of 12 h, which implies a turnover rate of 30,000 +/- 7500 receptors/h per cell. With progression to confluency, the Bmax value decreased up to 0.28 +/- 0.08 pmol/10(6) cells without significant change in Kd value. When cells were grown at a low density in serum-free conditions, the number of [3H]PMD binding sites gradually increased 1.9-fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP-sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.     

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of ¹²⁵I-IGF-I and [³H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of ¹²⁵I-IGF-I across HUVE cell monolayers was not significantly different to that of [³H]inulin, a paracellular probe. Moreover, ¹²⁵I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit ¹²⁵I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997. © 1997 Wiley-Liss, Inc.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.     

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39.

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of ³H-labelled [DAla²]-[Dleu⁵]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control. On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [³H]dihydromorphine and [³H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To wval;uate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 μM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligan-receptor recognition. Whether this can offer a satisfactory explanation for our obervations remains to be established.     

A serum-free,chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

Summary A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham’s F12 and Dulbecco’s modified Eagle’s medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC),l-thyroxine (T₄), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grow in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T₄, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Cholinergic and adrenergic receptors on mouse cardiocytes in vitro

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2–14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via α- and β-adrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [³H]Alprenolol, a potent β-adrenergic receptor antagonist, and [³H]quinuclidinyl benzilate ([³H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize β-adrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave ～800 sites/μm², a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells.     

Culture of cardiac muscle cells in serum-free media

Cardiac muscle cells from neonatal rats have been cultured in completely defined serum-free media. The most successful system consists of precoating culture flasks with fibronectin at a concentration of 5 μg/cm² of surface area and adding fetuin and either dibutyryl cyclic AMP (db-cAMP), cholera toxin, epidermal growth factor or insulin plus dexamethasone to the medium. In order to define a serum- and a hormone or growth factor-free medium, cardiac muscle cells were grown in the presence of fibronectin, fetuin and db-cAMP for 4 days, after which time db-cAMP was omitted from the medium. Under these conditions the cells continue to maintain their differentiated morphology for at least 4 days thereafter. These morphological studies demonstrate that dissociated neonatal cardiac muscle cells are able to grow and differentiate in a chemically defined medium in the absence of animal serum.     

Effects of Dietary n-3 Fatty Acids on the Phospholipid Molecular Species of Monkey Brain

Y-79 human retinoblastoma cells grown in serum-free medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y-79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y-79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of approximately 3 fmol/10(6) cells (approximately 1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly-D-lysine and fibronectin and continued culture in serum-free medium. After 8 days in serum-free culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/10(6) cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15-17 days (7-9 days of Bt2cAMP treatment) expressing receptor levels of 15-20 fmol/10(6) cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y-79 (dY-79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM-09151-2 approximately spiperone greater than domperidone approximately (+)-butaclamol approximately fluphenazine greater than chlorpromazine greater than (-)-sulpiride greater than (+)-sulpiride greater than promethazine greater than (+)-SCH 23390 much greater than (-)-butaclamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.     

Characterization of Muscarinic Receptors: Type M2 (Subtype B) on Neuro-2A Neuroblastoma Cells

Abstract

The binding of the nonselective muscarinic antagonist, [³H]N-methylscopolamine (NMS) to a mouse neuroblastoma cell line (Neuro-2A) and its coupling to the inhibition of adenylate cyclase were characterized. Specific [³H]NMS binding to membrane preparations was rapid, saturable, and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand. Competition experiments with the muscarinic drugs pirenzepine, AF DX 116, dicyclomine and atropine revealed that the muscarinic receptors present on these cells are predominantly of a single class, subtype B (M2). In addition, agonist binding demonstrated existence of a GTP-sensitive high affinity binding state of the receptors. Coupling of these muscarinic receptors to the adenylate cyclase system was investigated using the muscarinic agonist carbachol which was able to inhibit the prostaglandin (PGE₁)-stimulated activation of adenylate cyclase. The agonist carbachol did not stimulate the formation of IP₃ above basal levels, which indicated that the receptors are not coupled to phosphatidylinositol metabolism. In conclusion, we show that possessing predominantly one subtype of muscarinic receptor, the Neuro-2A cells provide a useful model for the investigation of the heterogeneity of muscarinic receptors and the relationship of subtype to the coupling of different effectors.     

Different sedimentation properties of agonist- and antagonist-labelled platelet alpha2 adrenergic receptors

Alpha₂ adrenergic receptors were solubilized from human platelet particulate preparations with digitonin. The solubilized alpha₂ receptors retained the essential binding specificity characteristics of the membrane-bound receptors. The alpha₂ receptors could be labelled in platelet membranes with either agonist ([³H]epinephrine) or antagonist ([³H]yohimbine) radioligands. When these membranes were solubilized with digitonin and centrifuged on sucrose density gradients, the sedimentation coefficient of the agonist-labelled receptor (14.6S) was greater than that of the antagonist-labelled receptor (12.9S). This observation may provide insight into the mechanism of adenylate cyclase inhibition by alpha₂ adrenergic receptors.