The ability of calcium to change cyclic AMP from a stimulator to an inhibitor to thymic lymphoblast proliferation

Calcium is a major regulator of thymic lymphoblast proliferation in vivo and in vitro. The proliferative activity of the lymphoblasts in thymic lymphocyte (thymocyte) populations in vitro is both constant and low in the presence of calcium concentrations between 0 and 1.0 mM, but higher concentrations increase proliferation by an endogenous cyclic AMP-mediated promotion of the initiation of DNA synthesis. Lower concentrations (10^?7 to 10^?5 M) of exogenous cyclic AMP (but not 5′-AMP) stimulate lymphoblast proliferation in a low-calcium (0.5 mM) medium, but higher concentrations do not. However, all exogenous cyclic AMP concentrations between 10^?7 and 10^?3 M (but again not 5′-AMP) block the stimulation of lymphoblast proliferation in a high-calcium (1.5 mM) medium. Exogenous cyclic AMP does not prevent calcium from “activating” lymphoblasts, but it reversibly blocks the reaction responsible for the initiation of DNA synthesis in these stimulated cells. Finally, cyclic AMP's inhibitory action, in contrast to its stimulatory action in low-calcium medium, is not specific for the cyclic nucleotide since a low, non-mitogenic concentration of cyclic GMP also prevents calcium from stimulating DNA synthesis and cell proliferation.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

The tumor promoter 12-0-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca²⁺. In contrast, the induction in cells grown in low calcium medium was 2–10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca²⁺ medium were switched to 1.2 mM Ca²⁺, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca²⁺ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.     

Stimulation of liver cell DNA synthesis by oncomodulin, an MW 11 500 calcium-binding protein from hepatoma

Raising the calcium concentration, or adding the tumor-specific calcium-binding protein oncomodulin (but not a similar, calcium-binding protein such as skeletal muscle parvalbumin) stimulated DNA synthesis in non-neoplastic T51B rat liver cells, whose DNA-synthetic activity had been reduced by incubation in medium containing 0.02 mM calcium instead of the usual 1.8 mM calcium. A calcium: oncomodulin complex was probably the actual stimulator, because oncomodulin action was blocked by further reducing the ionic calcium concentration in the already calcium-deficient medium with EGTA. Oncomodulin was also able to stimulate DNA synthesis in T51B cell cultures, whose response to calcium addition had been blocked by trifluoperazine.     

Mitogen-induced T-lymphocyte proliferation: a reappraisal of the early requirement of extracellular ionized calcium.

Mitogen-induced DNA synthesis in lymphocyte cultures requires an extracellular calcium concentration of 3 × 10^?6M or higher. When cultures of human or mouse lymphocytes were incubated with T-cell mitogens for the first 12 hours in a medium with about 3 × 10^?6M calcium ion concentration, and then the normal calcium concentration was restored, the induction of DNA synthesis in the cultures was salvaged, but it started 10–16 hours later than in control cultures. Lipopolysaccharide-induced thymidine incorporation in mouse spleen cell cultures responded to this experimental design in a more complex way. - These results support the idea that calcium ions are specifically needed for one or more of the very early steps in mitogenic activation of T-lymphocytes.     

Planarian regeneration: in vivo and in vitro effects of calcium and calmodulin on DNA synthesis

Regeneration does not occur when planarians are grown in Ca2+-free medium. The possible effect of calcium upon DNA synthesis was therefore studied using cultured planarian cells and regenerating planarian fragments. In the cultures, DNA synthesis was Ca2+-dependent and required a minimum of 10(-6) M Ca2+ in the medium. It was gradually decreased in cells grown in Ca2+-free medium. Addition of Ca2+ to these cultures raised DNA synthesis. The time lag between addition of Ca2+ and stimulation of DNA synthesis varied with culture age. The triggering effect of Ca2+ was amplified by ionophore A 23187. A calcium binding protein, ram testis calmodulin, intensified the stimulatory effect of calcium, but EGTA blocked this effect. In the presence of trifluoperazine (TFP), DNA synthesis was not stimulated by Ca2+. This inhibition by TFP was overcome by adding calmodulin to the medium. Ca2+ therefore triggered DNA synthesis in vitro, and this role might have been potentiated by calmodulin. In vivo, DNA synthesis was shown to be dependent on the Ca2+ concentration in the medium in which intact or regenerating planarians were grown. In 12-h regenerates, the Ca2+ concentration in the medium was no longer critical. Total calcium content decreased just after sectioning until completion of healing (at 6 h) and then rose significantly to a peak at 12 h which coincided with the first peak of DNA synthesis. The calmodulin content gradually diminished during the first 6 h after sectioning. After a transient rise at 12 h, calmodulin content further decreased until 48 h. The results demonstrate the crucial role of Ca2+ in triggering DNA synthesis in planarian cells in vitro and in regenerating fragments. Calmodulin, whose concentration is very low in planarians compared to vertebrates, might help to induce the first peak of DNA synthesis at 12 h after sectioning, but is probably not the main Ca2+-binding protein involved in the regeneration process.     

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca²⁺ and Mg²⁺ concentrations were high. Growth in medium high in Ca²⁺, Mg²⁺, and substrate (i.e., 150 mM acetate, 150 mM methanol, or 40 mM trimethylamine) converted pseudosarcinal aggregates to individual cocci. In such media, aggregates separated into individual cells which continued to grow exclusively as single cells during subsequent transfers. Conversion of single cells back to aggregates was complicated, because conditions which supported the aggregated morphology (e.g., low calcium or magnesium concentration) caused lysis of coccoid inocula. We recovered aggregates from coccoid cells by inoculating serial dilutions into medium high in calcium and magnesium. Cells from very dilute inocula grew into aggregates which disaggregated on continued incubation. However, timely transfer of the aggregates to medium low in calcium, magnesium, and catabolic substrates allowed continued growth as aggregates. We demonstrated the activity of the enzyme (disaggregatase) which caused the dispersion of aggregates into individual cells; disaggregatase was produced not only during disaggregation but also in growing cultures of single cells. Uronic acids, the monomeric constituents of the Methanosarcina matrix, were also produced during disaggregation and during growth as coccoids.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

The different actions of normal and supranormal calcium concentrations on the proliferation of BALB/c 3T3 mouse cells

Summary In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. However, the development of such stimulated, calcium-deprived cells stops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later. Issued as NRCC No. 15371.     

A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca²⁺ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 × 10⁴?2 × 10⁵ infectious centers per microgram of PM2 DNA was developed.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.     

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM · (Ca²⁺)₄ is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01–1 μ M) was 0.9 nM; the respective apparent dissoclation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1–50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by ¹²⁵I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM · (Ca²⁺)₄. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E · CaM) is at least 100-fold greater than the apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex, as judged from non-saturation ¹²⁵I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Effect of melanostatin and thyroliberin on the biosynthesis and release of dopamine by rat brain striatal P2 fractions

The effect of melanostatin (MIF) and thyroliberin (TRH) on dopamine biosynthesis and release has been studied in striatal P₂ fractions from rat brain. Dopamine synthesis was not affected by either MIF (10^?4M) or TRH (10^?4M) in the presence of 2 mM EDTA. In contrast, tyramine (10^?4M) and (+)-amphetamine (10^?4M) both stimulated dopamine biosynthesis, in a manner inversely proportional to the rate of dopamine biosynthesis, in the absence of drugs. MIF (10^?3M) or TRH (10^?3M) stimulated the release of dopamine in the presence of 1.25 mM calcium chloride but not in the presence of 2 mM EDTA. In contrast (+)-amphetamine 10^?4M) stimulated release of dopamine under both conditions. These results are discussed in relation to the hypothesis that MIF and TRH stimulate the release of dopamine from vesicular stores by a calcium dependent mechanism, whereas amphetamine stimulates release of dopamine from both vesicular stores and a cytoplasmic pool which regulates tyrosine hydroxylase activity.     

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with ³H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of ³H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of ¹⁴C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of ³H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous ³H-TdR from accurately reflecting the true level of DNA synthesis.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.     

Stimulation of hepatocyte DNA synthesis by neurotensin

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10^?10 M, and it was increased in a dose-dependent manner with maximal effects at 10^–8 M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor β. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter. © 1994 Wiley-Liss, Inc.     

Characterization of Acetylcholine-induced Increases in Cytosolic Free Calcium Concentration in Individual Rat Pancreatic β-cells

The intracellular free Ca²⁺ ion concentration ([Ca²⁺]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca²⁺]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca²⁺ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca²⁺]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca²⁺]i were rapid, transient and were blocked by atropine at 10^?6M. A brief exposure to 50 mM K⁺ resulted in a transient increase in [Ca²⁺]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10^?4M or 10^?3M) induced [Ca²⁺]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca²⁺ free medium, the source of the increase in [Ca²⁺]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca²⁺]i. These data strongly suggest that ACh exerted a direct action on [Ca²⁺]i in normal rat pancreatic β-cells and support a role for Ca²⁺ as a second messenger in the action of ACh.     

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Depletion of Mg²⁺ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by ³H-thymidine incorporation, and concomitant decreases in cellular K⁺ and Mg²⁺ and increases in Na⁺ and Ca²⁺. In cells grown in media containing 0.2 mM Ca²⁺, graded reduction of Mg²⁺ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg²⁺. Concomitantly, cell cations showed graded changes, Na⁺ increasing to 227%, K⁺ decreasing to 52.5%, Mg²⁺ decreasing to 57.5% and Ca²⁺ increasing to 153.5% of control. The effects of Mg²⁺ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca²⁺ concentration, the effects being larger at low Ca²⁺ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg²⁺ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg²⁺ depletion. The effects of Mg²⁺ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg²⁺ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg²⁺ in the coordinate control of growth and metabolism in animal cells.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.