bluuebunny/crossref_metadata_2025 huggingface dataset:
A reduction in the activity of the NA+, K+ ‐pump in dimethylsulfoxide‐treated friend erythroleukemia cells is not due to partial inactivation of the NA+, K+ ‐ATPase
Authors: Schaefer Andras , Munter Karl‐Heinz , Geck Peter , Koch Gebhard
Published: February 2005

Treatment of Friend‐erythroleukemia cells with 1.5% dimethylsulfoxide (DMSO) caused a decrease in ouabain sensitive 86Rb+‐uptake beginning six to seven hours after DMSO addition indicating a reduced function of the Na+, K+‐pump. However, analysis of the ouabain sensitive 86Rb+‐uptake after Na+‐preloading of the cells as well as measurements on the Na+, K+‐ATPase activity in isolated membrane fragments revealed that no inhibition of the Na+, K+‐ATPase occurred during the first 12 hours. On the contrary the Na+, K+‐ATPase activity was initially enhanced and then returned to control levels during the early phase of induction by DMSO. On the other hand, 22Na+‐transport into DMSO‐treated cells was reduced similar to the ouabain sensitive 86Rb+ uptake in cells without Na+ preloading. The piretanide sensitive 86Rb+‐uptake, due to the Na+, K+, 2Cl− cotransport system was inhibited after seven hours exposure to DMSO. Some three hours after DMSO addition the incorporation of 35S‐methionine into proteins began to decrease, which was accompanied with or followed by a reduction in the methionine uptake of DMSO treated cells. Membrane‐potential‐dependent tetraphenylphos‐phonium cation uptake was not altered relative to the controls in the first 12 hours following DMSO addition. These results suggest that the reduced activity of the Na+, K+‐pump in Friend cells after DMSO exposure is not due to inhibition of the Na+, K+‐ATPase, but most probably due to a smaller Na+‐influx, which results from inhibition of Na+‐cotransport processes (amino acid uptake, Na+, K+, 2Cl− cotransport system).
In vitro stimulation of articular chondrocyte differentiated function by 1,25‐dihydroxycholecalciferol or 24R,25‐dihydroxycholecalciferol
Authors: Harmand M. F. , Thomasset M. , Rouais F. , Ducassou D.
Published: February 2005

The effects of 1,25‐dihydroxycholecalciferol (1,25‐(OH)2D3) (10−13M‐10−8 M) and 24R,25‐dihydroxycholecalciferol (24R,25‐(OH)2D3) (10−12M‐10−7M) on cell proliferation and proteoglycan deposition were examined in our newly developed multilayer culture system for rabbit and human articular chondrocytes. The cells are embedded in an extracellular matrix similar to that seen in vivo and maintain their in vivo phenotype. We extracted and purified native proteoglycans and degraded material from three culture compartments: the medium, intercellular matrix, and cells. Proteoglycan synthesis and deposition were analyzed by measuring 35SO4 incorporation, hexuronic acid, and galactose contents. In both rabbit and human chondrocyte cultures, chronic 1,25‐(OH)2D3 treatment inhibited chondrocyte proliferation and stimulated proteoglycan synthesis and accumulation in the three compartments at 10−12‐10−8M. maximal effect was at 10−10M. Cell proliferation was reduced by 55% and the content of hexuronic acid (or galactose) was increased to about three times that of controls in all compartments. 1,25‐(OH)2D3 did not alter the proteoglycan composition. Chronic 24R,25‐(OH)2D3 treatment induced comparable effects with a maximum at 10−8M. When human dermal fibroblasts were treated as above both vitamin D metabolites increase mitosis. 1,25‐(OH)2D3 mainly reduced the pericellular deposition of proteoglycans, while 24R,25‐(OH)2D3 appeared to reduce their synthesis and deposition in both medium and pericellular compartments. These results suggest that both 1,25‐(OH)2D3 and 24R,25‐(OH)2D3 act specifically on articular chondrocytes to promote phenotype expression.
The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells
Authors: Yajima Yukiko , Saito Toshikazu
Published: February 2005

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.
Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia—prostaglandin E2
Authors: Rudland Philip S. , Twiston Davies Anna C. , Tsao Sai‐Wah
Published: February 2005

Cell strains and cell lines rat mammary (Rama) 350–353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cells on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100‐fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (>100 ng/mg/24 hr) of PGE; the fast‐sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10–100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2–4‐fold at concentrations (10–20 ng/ml) well below those released by the preadi‐pocytic stromal cells (20–100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2–4‐fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial‐like cell lines normally secrete moderate levels of PGE (10–100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PC's nor by PG‐receptor antagonists.
Evolution of cAMP phosphodiesterase activity in cultured myometrial cells: Effects of steroids and of successive subcultures
Authors: Vallet‐Strouve C. , Ferre F. , Breuiller M.
Published: February 2005

Cyclic AMP phosphodiesterase (PDE) activity was characterized in culture of ewe myometrial cells and its sensitivity to steroid hormones was tested. Cultured myometrial cells were maintained from the first to the 20th subculture in the presence of 2% of serum in a medium supplemented with 1 μM of insulin. It was found that myometrial cells possess a PDE activity with atypical kinetics. The nonlinear responses in Lineweaver‐Burke plots suggest the presence of high‐ and low‐affinity PDE activities. In cell culture, apparent Km values were similar to those obtained from the original myometrium. Vmax values increased with successive subcultures, revealing an increase in the capacity of the cells to degrade cAMP; in parallel, the growth rate decreased. The PDE specific activity in cultured myometrial cells was inhibited by estra‐diol or progesterone. When added together, no synergistic effect was obtained. The rate of inhibition for both steroids was constant during successive passages for both low‐ and high‐affinity conditions. Results obtained in myometrial cell long‐term culture were compatible with reports in other species in vivo. Considering the role of cAMP in the regulation of uterine functions, subcultured myometrial cells provided us a useful experimental system with which to study the cAMP metabolism process.
The intracellular pH of quiescent and proliferating human and rat thymic lymphocytes
Authors: Grinstein S. , Cohen S. , Lederman H. M. , Gelfand E. W.
Published: February 2005

We compared the cytoplasmic pH (pHi) of quiescent and actively cycling thymic lymphocytes. Human and rat thymocyte suspensions were fractionated by centrifugation on one step albumin density gradients. The pellet was composed of small, quiescent cells and the interphase contained mostly larger, actively cycling cells with a high proliferation index. When measured using [14C]‐dimethyloxazolidinedione (DMO), pHi in the large cells of both species was approximately 0.15 units more alkaline than in the small cells. However, these differences were not detectable when pHi was measured with carboxylated fluorescein derivatives generated in situ by cytoplasmic enzymes. This apparent discrepancy can be explained by compartmentation of DMO, which accumulates in the alkaline mitochondrial matrix. Comparison of the mitochondrial content of quiescent and cycling thymocytes by several methods showed that the latter contained over 2.5‐fold more mitochondria per unit cell volume. Assuming a constant intramitochondrial pH, this difference can account for the observed accumulation of DMO (i.e., apparent cytoplasmic alkalinity) in the actively proliferating cells. Therefore, no evidence was found for the existence of differences in pHi between quiescent and proliferating lymphocytes. Moreover, caution must be exercised when comparing DMO partition data in cells with varying relative mitochondrial content.
Amino acid activation of amino acid transport system N early in primary cultures of rat hepatocytes
Authors: Weissbach Lawrence , Kilberg Michael S.
Published: February 2005

System N, a neutral amino acid transport system characterized in rat hepatocytes, shows significant changes in activity during the first 24 hr of primary culture (Weissbach, L., Handlogten, M.E., Christensen, H.N., and Kilberg, M.S. [1982] J. Biol. Chem. 257:12006–12011). Experiments presented in the present report demonstrate that during the first 12 hr of primary culture System N can be stimulated by individual amino acids in the culture medium by a cycloheximide‐insensitive mechanism. This enhanced activity results from an elevation in the Vmax of the transport system, and the magnitude of the increase is related to the concentration of the amino acid in the culture medium. Nonsubstrates as well as substrates of System N are effective, and trans‐stimulation does not appear to play a role in this phenomena. Transport by Systems ASC, Gly, and L is enhanced by the presence of amino acids in the culture medium, but these systems are significantly less sensitive than System N. The results suggest that amino acids act at a posttranslational step to activate System N activity.
2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin: Examination of biochemical effects involved in the proliferation and differentiation of XB cells
Authors: Knutson Joyce C. , Poland Alan
Published: February 2005

XB, a cell line derived from a mouse teratoma, differentiates into stratified squamous epithelium when incubated with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). To examine the possible biochemical mediators of this response, we compared the effects produced by TCDD to those elicited by other compounds which stimulate epidermal proliferation and/or differentiation in mice. XB/3T3 cultures keratinize when incubated with cholera toxin, epidermal growth factor (EGF), or TCDD, but not 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA). Incubation of XB cells with TCDD (10−9M) for 48 hours produces a 20% increase in thymidine incorporation, a response which is neither as large nor as rapid as that produced by cholera toxin, TPA, or EGF. Although both cholera toxin and TCDD stimulate differentiation and thymidine incorporation in XB/3T3 cultures, cholera toxin increases cAMP 30‐fold in these cells, while TCDD does not affect cAMP accumulation at any of the times studies (15 min to 120 hours). Inhibitors of arachidonic acid metabolism, which block epidermal proliferative responses to TPA in vivo, do not prevent the differentiation of XB cell in response to TCDD. In XB/3T3 cultures, TPA stimulates arachidonic acid release at all times tested (1, 6, and 24 hours) and increases the incorporation of 32Pi into total phospholipids and phosphatidylcholine after 3 hours. In contrast, TCDD affects neither arachidonic acid release nor the turnover of phosphatidylinositol or phosphatidylcholine at any of the times tested. Although we examined biochemical effects which have been suggested as part of the mechanism of TCDD and which are produced by other epidermal proliferative compounds in XB cells, no mediator of the TCDD‐produced differentiation of XB/3T3 cultures was observed.
Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation
Authors: Lauf P. K. , Zeidler R. B. , Kim H. D.
Published: February 2005

Influx of the K+ analogue Rb+ was measured through the ouabain‐sensitive Na+/K+ pump and the ouabain‐insensitive “leak” pathways in Cl− or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7‐day‐old piglets. In reticulocytes, Rb+ influxes by the two pathways were of about equal magnitude in Cl− (13 and 10 mmoles/liter cells × hr) and at least 25‐fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na + media, a portion of the ouabain‐insensitive “leak” flux of Rb+ was Cl− dependent (Rb+Cl− transport) as NO replacement reduced Rb+ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N‐ethylmaleimide (NEM) stimulated Rb+Cl− transport about twofold in reticulocytes and up to 13‐fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain‐sensitive Rb+ pump and ouabain‐insensitive Rb+Cl− influx were lost. In contrast, the NEM‐stimulated Rb+Cl− transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb+Cl− transport. Although the experimental variability precluded a full assessment of significant changes in the small Na+/K+(Rb+) pump and Rb+Cl− fluxes in mature pig red cells kept for the same time period in vitro, Rb+ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na+/K+ pump and Cl−‐dependent K+ fluxes in vivo.
Comparison of intracellular and extracellular mitogenic activity
Authors: Gajdusek Corinne M. , Schwartz Stephen M.
Published: February 2005

Endothelial cell‐derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat‐stable and trypsin‐sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen‐secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56°C) and were not inactivated by trypsin. Similar to platelet‐derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.
Maturation of human promyelocytic leukemia cells induced by nicotinamide: Evidence of a regulatory role for ADP‐ribosylation of chromosomal proteins
Authors: Lucas Diane L. , Tanuma Sei‐Ichi , Davies Peter J. A. , Wright Daniel G. , Johnson George S.
Published: February 2005

We have studied the role of ADP‐ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL‐60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP‐ribose)n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP‐ribose)n synthetase was decreased in HL‐60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP‐ribose)n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N′‐methylnicotinamide (N′‐Met‐NAm), agents which decrease ADP‐ribosylation. Treatment of HL‐60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N′‐Met‐NAm was more potent than NAm in inducting maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N′‐Met‐NAm also potentiated the maturation of HL‐60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose‐response curve for RA; maturation was observed at 10−11 M RA in the presence of either 2 mM NAm or 0.2 mM N′‐Met‐NAm while 10−9 M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N′‐Met‐NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N′‐Met‐NAm decreased by about 75% the amount of endogenous (ADP‐ribose)n in a selected fraction of chromosomal proteins which included histone H1 and the nonhistone high mobility group proteins. The results of this study support the concept that ADP‐ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.
Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells
Authors: Boman Bruce M. , Zschunke Michael A. , Scott Robert E.
Published: February 2005

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP‐dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP‐dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP‐dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP‐dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.
β‐Adrenergic stimulation inhibits calcium efflux and alters the morphology of cultured arterial muscle cells
Authors: Smith Jeffrey Bingham
Published: February 2005

β‐Adrenoreceptor stimulation with isoproterenol (IP) rapidly and reversibly rounded and arborized smooth muscle cells (SMC) cultured from rat aorta. The arborized SMC remained firmly attached to the substratum via a network of long, dendritelike processes. Arborization of the SMC correlated closely with increases in cellular cAMP produced by IP and a variety of other compounds. The intracellular calcium antagonist 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate (TMB‐8) also rounded and arborized the SMC. Antitubulin compounds potently blocked arborization by IP, dibutyryl cAMP, and TMB‐8. The release of cell‐bound 45Ca2+ was followed in the presence and absence of IP. IP strikingly increased the amount of 45Ca2+ that remained cell bound between 20 and 120 min Propranolol and colchicine prevented IP from inhibiting the release of cell‐bound 45Ca2+. These results suggest that the modulation of Ca2+ transport is involved in the arborization of cultured SMC by cAMP.
Recovery of cyclic nucleotide regulation in protein‐kinase‐defective adrenal cells through somatic cell fusion
Authors: Schimmer Bernard P. , Horney Sandra J. , Williams Shirley A. , Aitchison William A. , Doherty Paul J.
Published: February 2005

A mutant cell line (designated Kin‐8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth‐inhibition by 8‐bromoadenosine 3′, 5′‐monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin‐8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP‐dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin‐8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP‐dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin‐8 × C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin‐8 × C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50 values for 8BrcAMP. In addition, Kin‐8 × C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effect of 8BrcAMP on the steroidogenic activity and morphology of Kin‐8 × C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP‐responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP‐dependent protein kinase for Kin‐8 cells and led to the recovery of a cAMP‐responsive adrenal phenotype. These results provide additional evidence in support of an obligatory role for cAMP‐dependent protein kinase in the regulation of adrenal steroridogenesis, cell division, and cell shape.
Effects of cystic fibrosis serum on calcium influx and secretion using isolated tracheal epithelial cells
Authors: Wooten M. W. , Rudick M. J. , Rudick V. L. , Kramer R. I.
Published: February 2005

Hamster trachea epithelial (HTE) cells were shown to respond to 20% Cystic fibrosis serum (CFS) by secreting twice as much protein as in the presence of 20% normal human serum (NHS). Seum from obligate heterozygotes (HHS) produced an intermediate effect. A peak of Ca2+ entry into the HTE cells occurred about 30 min after exposure to 20% CFS, followed by a slow decline to basal levels. In contrast, 20% NHS did not cause an influx of Ca2+ and HHS produced an influx to about half that of CFS. Increasing concetrations (5‐30%) of pooled NHS had no effect on HTE cell Ca2+ uptake or secretion, but pooled CFS and HHS caused progressive increases in Ca2+ influx and protein secretion from 10 to 25% sera. The CFS‐induced Ca2+ influx and secretion were about twice those of HHS throughout the range of serum concentrations tested, suggesting the presence of a modulatory influence in HHS. When EGTA was used to chelate extracellular Ca2+ in the presence of CFS, Ca2+ influx was prevented and there was no stimulation of secretion. lonophore A23187 allowed Ca2+ entry into HTE cells in the presence or absence ofserum and a heightened level of secretory activity followed. The time course of Ca2+ influx under the influence of CFS was shown to correspond to the efflux of Na+ from the cells. Also verapamil, a Ca2+ channel blocking agent, inhibited CFS‐induced Ca2+ influx by 50% at 10−5M and prevented secretion. Thus, it appears that CFS, but not NHS, contains an agent which stimulates Ca2+ uptake into HTE cells by means of a Ca2+ channel and/or Na+‐Ca2+ exchange mechanism, and that increased intracellular Ca2+ levels then trigger secretion. The intermediate HTE cellr esponse to HHS suggests that half of the CFS stimulatory agent is present as would be expected in a gene dose effect, lending support for a agentic basis for CF.
Modulation of Ca2+‐mediated K+‐gating of erythrocyte ghosts by external Ca‐EGTA
Authors: Benjamin A. M. , Quastel D. M. J.
Published: February 2005

Using 86RB+ as a marker for K+ permeability, we find that extracellular Ca‐EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and “buffered” Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca‐EGTA at 0.2–0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca‐EGTA (up to 1–2 mM) diminish the flux rate. External Ca‐EDTA or Mg‐EDTA can substitute for Ca‐EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg‐EDTA does not substitute for internal Ca‐EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca‐EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca‐EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1‐propranolol (25 μM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca‐EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+‐gate at the inner surface of the membrane. At external concentrations of Ca‐EGTA above 1–2 mM, 86Rb+ flux rate again rises with increase of Ca‐EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1‐propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.
The in vitro cell culture age and cell density of articular chondrocytes alter sulfated‐proteoglycan biosynthesis
Authors: Malemud Charles J. , Papay Robert S.
Published: February 2005

The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated‐proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5‐7 d), and high density (HD, 12‐15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]‐thymidine. LD cultures incorporated 35SO4 into sulfated‐proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated‐proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCI density gradients and by elution profiles on Sepharose CL‐2B. Chondrocytes deposited into the extracellular matrix (cell‐associated fraction) 35SO4‐labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4‐labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell‐associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.
β adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation
Authors: Homburger V. , Pantaloni C. , Lucas M. , Gozlan H. , Bockaert J.
Published: February 2005

C6 glioma cells possess β adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br‐AAM‐pindolol), a β adrenergic antagonist. With 1 μM Br‐AAM‐pindolol, more than 80% of β adrenergic receptors, labeled by (3H)‐dihydroalprenolol ((3H)‐DHA), were blocked. After this blockade, new β adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of β adrenergic receptors was barely detectable. Cycloheximide (1 μg/ml) inhibited cell growth as well as reappearance of β adrenergic receptors. A 90% loss of β adrenergic receptors in C6 glioma cells was obtained after down‐regulation for 15 h with 10 μM isoproterenol, a β adrenergic agonist. After removal of the agonist, recovery of β‐adrenergic‐sensitive adenylate cyclase was complete within 2 to 3 days, whereas β adrenergic receptors reached 90% of control value within 6 days. The half‐life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br‐AAM‐pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked β adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of β adrenergic receptors. The recovery of both biological response and β adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 μg/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of β adrenergic receptors in C6 glioma cells, following isoproterenol‐induced down regulation, was not due to synthesis of new receptors but to recycling of the β adrenergic receptors.
Regulation of angiotensin I‐converting enzyme activity in serially cultivated bovine endothelial cells
Authors: Rosen Eliot M. , Noveral James P. , Mueller Stephen N. , Levine Elliot M.
Published: February 2005

Angiotensin I‐converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.
Modulation of beta‐adrenergic response in rat brain astrocytes by serum and hormones
Authors: Wu Doris K. , Morrison Richard S. , de Vellis Jean
Published: February 2005

Purified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta‐adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum‐free medium plus hydrocortisone, putrescine, prostaglandin F2α, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum‐supplemented medium, astrocytes grown in the chemically defined medium were nonresponsiveto isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta‐adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta‐adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum‐supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta‐adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta‐adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta‐adrenergic response.
Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities
Authors: Zipori Dov , Duksin Dan , Tamir Merana , Argaman Anat , Toledo Jeki , Malik Zvi
Published: February 2005

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA‐1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA‐13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA‐2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage‐colony‐stimulating factor, CSF‐1. MBA‐2 and to a lesser extent, MBA‐13, produced an additional activity resistant to anti‐CSF‐1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA‐2 cell line (MBA‐2.1 and MBA‐2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC‐11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.
Regulation of thymidylate synthase enzyme synthesis in 5‐fluorodeoxyuridine‐resistant mouse fibroblasts during the transition from the resting to growing state
Authors: Jenh Chung‐Her , Rao Lakshmi G. , Johnson Lee F.
Published: February 2005

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth‐stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5‐fluorodeoxyuridine (5‐FdUrd)‐resistant cell line (LU3‐7) that overproduces TS and its mRNA about 50–100‐fold. In this paper we show that the LU3‐7 cells were able to rest in the GO state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10% the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulselabeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8–9‐fold by 25 hr following serum stimulation. The half‐life of TS in growing LU3‐7 cells was measured in a pulse‐chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3–7 cells and in the parental 3T6 cells, the LU3–7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.
Subpopulations of fibroblasts from mouse skeletal muscle defined by clonal variation for 5′ nucleotidase expression
Authors: Turker Mitchell S. , Ogburn Charles E. , Martin George M.
Published: February 2005

A primary cloning technique has been employed for the isolation of nine spontaneously transformed cell lines from mouse skeletal muscle. Four of these lines were isolated after selection for partial resistance to the purine (adenine) analog 2′6′diaminopurine and five were isolated from non‐selected control dishes. Four of the nonselected lines and three of the selected lines demonstrated a fibroblastoid morphology in vitro. The other two cell lines (one from each group) were epithelioid. Two of the three selected fibroblastoid lines were found to contain significant quantities of the enzyme 5′nucleotidase (EC3.1.3.5), whereas the four nonselected fibroblastlike lines, one selected fibroblastlike line, and the two epithelioid lines did not. In the two cell lines expressing 5′nucleotidase activity, this expression was stable in the absence of selective pressure. Histochemical staining of mouse skeletal muscle for 5′nucleotidase activity demonstrated positive staining in the cells of small blood vessels and in a subset of the connective tissue cells. The bulk of the skeletal muscle tissue, however, had no detectable 5′nucleotidase activity. We propose that the two cultivatable types of fibroblastoid cell lines represent distinct classes of fibroblastlike cells in vivo, reflecting alternative states of stable cellular differentiation involving 5′nucleotidase expression.
Proliferation and differentiation in culture of mast cell progenitors derived from mast cell‐deficient mice of genotype W/Wv
Authors: Suda Toshio , Suda Junko , Spicer Samuel S. , Ogawa Makio
Published: February 2005

Mice of genotype W/Wv have less than 1% of normal mast cells in the skin, stomach, and cecum. In order to further clarify the mechanism of this deficiency, we studied committed mast cell progenitors and multipotent progenitors, which are capable of mast cell differentiation in clonal culture. The relative concentration of mast cell progenitors in the bone marrow, spleen, and peripheral blood of W/Wv mice was similar to that of +/+ mice. However, the cellularity of the marrows of W/Wv mice was 54% of that of their normal littermates. Identification of mast cells was established by metachromatic staining with toluidine blue, transmission electron microscopy, and demonstration of membrane receptors for immunoglobulin E. The time course of colony formation and the morphology of W/Wv mast cell colonies in culture was identical to that of normal littermates. The percentages of mast cells in individual multi‐lineage colonies were extremely variable. The histamine content of mast cells derived from W/Wv mice was similar to that of mast cells from +/+ mice. These studies demonstrated the normal capacity for differentiation and proliferation in culture of mast cell progenitors from W/Wv mice.
Linear relationship of phlorizin‐binding capacity and hexose uptake during differentiation in a clone of LLC‐PK1 cells
Authors: Amsler Kurt , Cook John S.
Published: February 2005

With a clone of (Cl 4) of LLC‐PK cells, which develop a high capacity for Na+‐dependent hexose uptake over time (days) in culture, we show that increasing uptake capacity is paralleled by an increase in the number of phlorizin‐binding sites in the population. The linear relationship between binding and hexose transport is the same whether the cells differentiate spontaneously or are induced by either methylisobutylxanthine or hexamethylene bisacet‐amide. The constancy of the relationship suggests that the primary factor in transport development is the number of transporters in the cells rather than other possible factors like a change in membrane potential or decreased efflux. The Kd for phlorizin binding is .08 ± .04 μM, and corresponds to Ki of 0.10 μM for transport inhibition. The turnover number of the transporter is estimated to be 170 ± 40 molecules per second of α‐methyl glucoside.
Intracellular calcium redistribution and its relationship to fMet‐Leu‐Phe, leukotriene B4, and phorbol ester induced rabbit neutrophil degranulation
Authors: Naccache P. H. , Molski T. F. P. , Borgeat P. , Sha'afi R. I.
Published: February 2005

The addition of low concentrations (<10−7 M) of the calcium ionophore A23187 to rabbit neutrophils re'eases the intracellular pool of calcium previously shown in radioactive steady‐state and chlortetracycline fluorescence studies to be mobilized by chemotactic factors. A23187 at these concentrations elicits no functional responses from these cells. However, A23187, added before chemotactic factors such as fMet‐Leu‐Phe and leukotriene B4, inhibits the ability of the latter stimuli to induce, in the presence of cytochalasin B, an exocytotic release of the neutrophil's cytoplasmic granules. These results imply that the chemotactic‐factor‐induced release of intracellular calcium is a necessary event for the optimal activation of the neutrophils. Phorbol esterinduced neutrophil degranulation on the other hand is unaffected by exposure to A23187, thereby completely dissociating its mechanism of action from rises in cytoplasmic free calcium.
Increased thermoresistance developed during growth of small multicellular spheroids
Authors: Wigle Jeffrey C. , Sutherland Robert M.
Published: February 2005

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (∼ 70 μm diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45°C for periods ≤ 2 hr and at 42.5°C for ≤ 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of (1) the extent of intercellular contact in spheroids of different cellular multiplicities, (2) differences in membrane damage influenced by trypsin heat treatment sequence, and (3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.
Adaptive regulation of neutral amino acid transport system A in rat H4 hepatoma cells
Authors: Kilberg Michael S. , Han He‐Ping , Barber Ellen F. , Chiles Thomas C.
Published: February 2005

Substrate regulation of System A transport activity in rat H4 hepatoma cells is described. The uptake of several amino acids was tested in the presence of system‐specific inhibitors. System A activity was increased in a RNA‐ and protein synthesis‐dependent manner by amino acid deprivation of the cells (adaptive regulation), whereas transport by Systems ASC, N, y+, and L was unaffected. Unlike human fibroblasts, the H4 cells did not require serum to exhibit the depression of System A. At cell densities between 88 × 103 and 180 × 103 cells/cm2, the degree of adaptive regulation was inversely related to cell density. Both transport of AIB and adaptive regulation of System A were nearly abolished if either K+ or Li+ was substituted for Na+ in the medium. The presence of cycloheximide or tunicamycin blocked further increases in starvation‐induced activity within 1 hr of addition, suggesting the involvement of a plasma membrane glycoprotein. In contrast, if the medium was supplemented with actinomycin after the stimulation of System A had begun, the activity continued to increase for an additional 2 hr before being slowed by the inhibitor. The contributions of trans‐inhibition and repression to the amino acid‐induced decay of System A activity were estimated for several representative amino acids. In general, the System A activity in normal rat hepatocytes was much less sensitive to trans‐inhibition than the corresponding activity in H4 hepatoma cells. The half‐life values for the amino acid‐dependent decay of System A ranged from 0.5 to 2.0 hr.