Influence of increased extracellular calcium concentration and donor age on density-dependent growth inhibition of human fibroblasts

Elevated calcium chloride concentration [( CaCl2]) has been shown to increase saturation density for an established mouse fibroblast line and for human fetal lung fibroblasts (WI-38). In order to examine the effect of increased [CaCl2] on human fibroblasts from donors of varying age, fibroblasts were grown in medium (basal level of 1.8 mM CaCl2) supplemented with fetal bovine serum (FBS) until confluent. Compared to controls in basal medium, newborn foreskin fibroblasts exposed to additional CaCl2 had a 110-450% increased cell yield that was independent of [CaCl2] within the range of an additional 1.5-5.0 mM. The effect was maintained over an eightfold range of FBS concentration. Initial growth rate was unaffected, but a prolongation of exponential phase occurred for cultures exposed to increased [CaCl2]. Confluent cultures refed medium with increased [CaCl2] were stimulated 5- to 10-fold more than cultures refed basal medium. An additional 2 mM CaCl2 resulted in a 210% increase for young adult-derived fibroblasts versus a 29% increase for old adult-derived fibroblasts (P less than 0.001). These data indicate that increased [CaCl2] decreases density-dependent growth inhibition of postnatal human dermal fibroblasts in vitro and that this effect is donor age dependent.     

Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes

The role of extracellular calcium (Caex) in modulating keratinocyte differentiation has been well documented, but its role in proliferation has been harder to define due to the confounding effect of terminal differentiation. Because strontium (Sr) does not induce terminal differentiation in murine keratinocytes but does mimic the stimulatory effect of Caex on DNA synthesis in chick fibroblasts, experiments were undertaken to determine if Sr could be used to separate the presumably opposing effects of Caex on the proliferation and differentiation of cultured human keratinocytes. In response to additions of SrCl2, keratinocytes in a serum-free hormone-supplemented basal medium containing 0.03 mM Ca showed a dose-dependent increase in day 7 cell yields. Cell yield in the optimal concentration of SrCl2 (1.8 mM) was approximately twice that obtained in any concentration of CaCl2. Maximally stimulatory additions of CaCl2 varied from 0.05 to 1.8 mM, but 0.03 and 0.05 mM additional CaCl2 always increased cell yield relative to unsupplemented controls. Keratinocytes grown in low levels of CaCl2 or any level of SrCl2 have minimal contact with each other regardless of cell density in contrast to the colonies of tightly apposed and stratified cells grown in 1.8 mM CaCl2. Transmission electron micrographs of vertically sectioned confluent cultures in low or high levels of SrCl2 or in low levels of CaCl2 revealed abundant ribosomes and keratin filaments but no stratification or desmosomes, while cultures in 1.8 mM CaCl2 were stratified with numerous desmosomes. These results suggest that Caex may separately stimulate keratinocyte proliferation and terminal differentiation and that Srex can substitute for Caex in the former but not the latter process.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Effects of ionophore A23187 and lanthanum on pepsinogen secretion from frog esophageal mucosa in vitro

The role of Ca2+ in the mediation of pepsinogen secretion from frog esophagus was investigated by means of ionophore A23187 and LaCl3. The esophageal mucosa from Asian bullfrog Rana tigerina was mounted in a double-chamber system to preserve its polarity and was incubated in a medium containing 1.5 mM CaCl2. Pepsinogen secreted was measured and expressed as % of total. The basal secretion averaged 3.5%/h. Bethanechol (25 microM), dibutyryl-cAMP (10 mM), ionophore A23187 (30 microM) and 3-isobutyl-1-methylxanthine (0.1 mM) increased the secretion to 8.7, 7.4, 7.1 and 6.8%, respectively. The stimulatory effect of bethanechol and of dibutyryl-cAMP were not affected by removing the exogenous Ca2+ with EGTA. The basal secretion was, however, reduced by 50% when Ca2+ in the incubation medium was lowered to 20 microM. At this low Ca2+ concentration, ionophore A23187 not only lost its stimulatory effect but also diminished the stimulation caused by bethanechol and dibutyryl-cAMP. While LaCl3 at 1 mM had no effect on basal and bethanechol-stimulated secretion, at 10 mM it abolished the stimulation evoked by bethanechol or dibutyryl-cAMP. The conclusions are: (1) both Ca2+ and cAMP are involved in the mediation of pepsinogen secretion from frog esophagus, (2) basal secretion is dependent on extracellular Ca2+, whereas bethanechol-stimulated secretion is not, (3) in the plasma membranes of peptic cells may exist a distinct Ca2+ pool (La3+-and ionophore A23187-sensitive) which is involved in the stimulated pepsinogen secretion.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

Modulation of WI-38 cell proliferation by elevated levels of CaCl2

Elevating the level of extracellular calcium (CaEx2+) increases the saturation density achieved by the normal human diploid cell line, WI-38, but does not change the growth rate. Day 7 cell yields remain unchanged when [CaEx2+] is between 0.5 mM and 3.0 mM, decrease when [CaEx2+] less than 0.5 mM, and increase when [CaEx2+] greater than 3.0 mM. Combining hydrocortisone with additional CaCl2 results in an additive effect on the saturation density relative to that obtained with each treatment separately. The stimulatory effect of elevated [CaCl2] is independent of serum concentration but is lost when WI-38 cells are grown in conditioned medium. Stimulation is recovered when conditioned medium is diluted with serum-free medium. In the case of young cultures grown in conditioned medium, stimulation can also be recovered when higher than usual levels of additional CaCl2 are used (2-3 mM). A glutamine supplementation to the conditioned medium potentiates cell response to elevated [CaCl2]. These results indicate that the loss of an enhanced saturation density when cells are grown in conditioned medium is not due to serum depletion but is more likely the effect of metabolites and/or nutrient depletion. When older or less vigorously growing cultures are grown in conditioned medium, additions of up to 3 mM CaCl2 only lead to inhibition, suggesting an age-related change in proliferative regulation. Elevated levels of CaEx2+ also enhance the proliferative response of quiescent monolayers to serum stimulation. This finding, along with the increase in saturation density of Ca2+-treated cultures, suggests that an elevated level of CaEx2+ affects cell entry into and exit from quiescence brought on by density-dependent inhibition.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.     

Growth stimulatory precipitates of Ca2+ and pyrophosphate

Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM. This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H-uridine, and 3H-choline uptake, and the incorporation of 3H-leucine and 3H-thymidine into trichloroacetic acid (TCA)-insoluble material. Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism. The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi. Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations), PPi (77% of anions), and Pi (23% of anions). Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi. We have distinguished cell surface-bound PPi from intracellular PPi by differential extraction. The quantity of surface-bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation. If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate-containing medium, the cells are not stimulated. These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi. PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I-platelet-derived growth factor to its specific cell-surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen-receptor systems. In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation. The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters.     

The effect of polyoma virus,serum factors,and dibutyryl cyclic AMP on dihydrofolate reductase synthesis,and the entry of quiescent cells into S phase

Three procedures were used to induce dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: (1) lytic infection with polyoma virus, (2) growth stimulation by replating cells at lower density in fresh cell culture medium, and (3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S-phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phase began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis. Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S-phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S-phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in Gl prior to either the induction of dihydrofolate reductase synthesis or the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.     

Umasking large and persistent reductions in proliferation rate of aging cells

Summary We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.     

Neuropeptides to replace serum in cryopreservation of mesenchymal stromal cells?

Background aimsThe therapeutic potential of human mesenchymal stromal cells (MSCs) has generated considerable interest in a wide variety of areas. MSC banking is feasible, but the optimal technique of cryopreservation remains to be determined.MethodsTo reduce dimethyl sulfoxide (DMSO) concentration in cryopreservation medium, DMSO was replaced with sucrose or trehalose. To increase cell survival and proliferation rates after thawing and to eliminate the need for fetal bovine serum (FBS), neuropeptides of the vasoactive intestinal peptide/glucose-dependent insulinotropic peptide/pituitary adenylate cyclase activating polypeptide family were added to the cryopreservation medium. Cell survival was analyzed by a trypan blue dye exclusion assay. Cell proliferation of cryopreserved MSCs was determined after 7 days of culture.ResultsNo significant differences in cell survival rates were detected between cryopreservation solutions with 5% and 10% DMSO, independently of the addition of trehalose or sucrose. Cell proliferation rates tended to be highest when MSCs were frozen in 5% DMSO + trehalose. FBS could be replaced by human albumin (HA) without loss in cell survival and proliferation potential. With FBS, the addition of neuropeptides could increase cell survival and proliferation rates. Without FBS or HA, cell survival and proliferation rates in the presence of neuropeptides were comparable to rates achieved with FBS or HA.ConclusionsClassic cryopreservation with 10% DMSO could be replaced by 5% DMSO + 30 mmol/L trehalose. FBS could be replaced by HA or neuropeptides without loss in cell survival and proliferation potential. The addition of neuropeptides in the cryopreservation medium containing FBS could increase the cell proliferation rate and consequently cellular output.     

Involvement of calcium ion in the stimulated shoot elongation of arrowhead tubers under anaerobic conditions

Shoot elongation of arrowhead (Sagittaria pygmaea Miq.) tubers was stimulated in anaerobic conditions. The anaerobic elongation was attributed to stimulation of cell elongation in the middle of the shoots. The anaerobic elongation of the shoots was severely inhibited by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The EGTA inhibition was completely nullified by exogenous CaCl2, which acts as an enhancer of anaerobic elongation. Moreover, calcium channel blockers, verapamil, diltiazem and LaCl3, inhibited the anaerobic elongation enhanced by CaCl2. These results showed that calcium plays an important role in stimulating the elongation in anaerobic conditions. Incorporation of 45Ca into the shoot tissues was measured to determine the involvement of calcium uptake in anaerobic elongation. Incorporation of 45Ca into the cell sap, which was collected from frozen and thawed shoots after thorough washing with LaCl3, was significantly stimulated in anaerobic conditions. Verapamil and diltiazem prevented the stimulation of 45Ca incorporation in anaerobic conditions. These results suggest that calcium uptake from the medium serves to enhance shoot elongation of arrowhead tubers under anaerobic conditions.     

Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.     

Ca2+]i independent mitogenesis in cultured human fibroblasts revealed by single cell microfluorimetry

A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of porcine fetal fibroblast nuclear transfer embryos

The present study examined the effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of fetal fibroblast nuclear transfer (NT) porcine embryos. Frozen-thawed and serum starved fetal fibroblasts were transferred into the perivitelline space of enucleated oocytes. Cell fusion and activation were induced simultaneously with electric pulses in 0.3 M mannitol-based medium containing 0.1 or 1.0 mM CaCl(2). Some fused embryos were further activated 1 hr after the fusion treatment by exposure to an electric pulse. The NT embryos were cultured in vitro for 6 days. Fusion and blastocyst formation rates were significantly (P<0.05) increased by increasing the Ca(2+) concentration from 0.1 mM (67.1 and 6.3%) to 1.0 mM (84.7 and 15.8%). However, no difference in the number of cells in blastocysts was observed between the two groups. A higher percentage of blastocyst was also observed when control oocytes were parthenogenetically activated in the presence of elevated Ca(2+) (19.3% vs. 32.4%, P<0.05). When the reconstituted oocytes were fused in the medium containing 1.0 mM CaCl(2), increasing the number of pulses from 2 to 3 or an additional activation treatment did not enhance the blastocyst formation rate or cell number in blastocysts. These results demonstrate that increasing the Ca(2+) concentration in the fusion/activation medium can enhance the fusion and blastocyst formation rates of fetal fibroblast NT porcine embryos without an additional activation treatment.     

Cell detachment mediated by hyaluronic acid

Exogenous hyaluronic acid (HA) added to a confluent monolayer of 3T3 BALB cells facilitates cell detachment which can be enhanced by gently pipetting. When HA is added to a cell culture with the cell inoculum, the cells are able to grow and form a confluent monolayer, but the cellular density is lower than in the control cultures, in a concentration-dependent way. This difference seems due to the ease of detachment promoted by HA on the cells near confluency. In fact only near confluency is the amount of the detached cells greater in the culture plates containing HA than in controls. Culture dishes containing substrate-attached material (SAM) left behind by the confluent 3T3 BALB cells have been prepared by removing the cells with different detaching agents. The SAM-containing dishes have been incubated in the presence of HA for 24 h and, after washing, were used for cell cultures. The cells grown on such HA-treated dishes show a very low density at confluency and in some cases are prevented from forming a confluent monolayer. When the SAM-containing dishes are treated with Streptomyces hyaluronidase, the effect of HA is abolished and the cells are able to grow normally. Among the chondroitin sulphates, only chondroitin sulphate C shows the same effects as HA, whereas A and B are ineffective.     

Evidence that lanthanum ions stimulate calcium inflow to isolated hepatocytes.

LaCl3 stimulated the initial rate of 45Ca2+ exchange measured under steady-state conditions in isolated liver cells. Cu2+ greater than La3+ = Fe3+ greater than Fe2+ = Zn2+ greater Ni2+ greater than Mn2+ also stimulated 45Ca2+ exchange. Compartmental analysis of 45Ca2+-exchange curves obtained in the presence or absence of La3+, and in the presence or absence of adrenaline, showed that the predominant effect of La3+ is to stimulate the inflow of Ca2+ to the cell from the medium. No evidence for an inhibition of Ca2+ outflow from the cell was obtained. In the presence of La3+, adrenaline caused no further stimulation of Ca2+ inflow to the cell. In the absence of adrenaline, La3+ increased the uptake of Ca2+ (measured by atomic-absorption spectroscopy) by isolated hepatocytes incubated at 1 degree C. The proposal that La3+ stimulates Ca2+ inflow to the liver cell by inducing a conformational change in the Ca2+-inflow transporter of the plasma membrane is briefly discussed.