Selective induction of tyrosine hydroxylase by cell-cell contact in bovine adrenal chromaffin cells is mimicked by plasma membranes

As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.     

Relationship between cell proliferation, chromatin template activity and accumulation of nuclear proteins

Trypsinization of confluent monolayers of WI-38 cells causes an extensive loss of nuclear proteins. The loss of nuclear proteins is restored only several hours after the cells have been replated at a lower density in 10% serum. When trypsinized fibroblasts are replated at a lower density in 10% serum, there is also a sustained progressive leading to DNA synthesis and cell division. If 0.3% serum is used instead of 10%, there is a modest increase in nuclear template activity, but not accumulation of nuclear proteins and no DNA synthesis or cell division.     

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10⁵ cells/cm², to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant (K_D) of 1 × 10 ⁹, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.     

Induction of pseudofoci and inhibition of density-mediated neoplastic transformation by PMA in NIH 3T3 cells after short-term exposures

Summary Depending on the precise conditions and cellular starting material, phorbol-13-myristate-12-acetate (PMA) can induce or suppress the transformation of NIH 3T3 cells. In sublines that do not undergo rapid transformation, exposure to PMA over the course of several weeks accelerated the process, while sublines that are primed for density-mediated transformation respond to PMA with a suppression of the process. This study examines the latter phenomenon. Within 1 h of exposure to 0.02μg/ml PMA, sparse cultures had undergone a morphological transition after which the cells appeared smaller and the processes thinner. These sublines exhibited a two-to sixfold increase in the saturation density achieved in 2% calf serum (CS). Phorbol ester analogs with hydrocarbon substitutions of 4 or more carbons at positions 12 and 13 of the phorbol nucleus had a similar effect as PMA on the saturation density. High concentrations of PMA (1μg/ml) induced the formation of cell aggregates (pseudofoci) that resembled transformed foci in their high local density, but unlike transformed foci, did not reinitiate focus formation if the cells were diluted and replated without PMA as secondary cultures. PMA inhibited the processes of neoplastic transformation and progression that occur readily in these NIH 3T3 sublines when they reach high cell density. I suggest that such changes occur because PMA abolishes the selection pressure at high densities that favors the transformation of some cells in heterogeneous populations. Induction of transformation by PMA (reported previously) occurs after much longer exposures in sublines that are relatively resistant to rapid density-mediated transformation. These results are discussed in the context of progressive state selection, a concept that has been developed to account for spontaneous transformation in this system.     

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.     

Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.     

Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.     

Effect of cell density on energy-dependent calcium uptake by Balb/c 3T3 membranes is independent of protein synthesis and attachment to substratum.

Membranes isolated from subconfluent cultures of Balb/c 3T3 cells have low energy-dependent calcium uptake activity. Replating confluent cells at low density results in a prompt fall of energy-dependent calcium uptake by membrane fractions. The level to which uptake activity falls is a function of the density at which the cells are plated (Moore and Pastan, '77b). To determine if regulation of energy-dependent uptake of calcium by membrane fractions is dependent upon attachment to a substrate and to further characterize conditions that regulate the process, we examined calcium uptake activity of membranes isolated from cells in suspension. With cells in suspension energy-dependent calcium uptake activity of isolated membranes falls promptly if cells are diluted to a low density (less than 10(5) cells/ml) and is a function of cell density. When cells in suspension at low cell densities are concentrated to high cell densities (greater than 2 x 10(6) cells/ml), calcium uptake activity of the isolated membrane fraction is increased as a function of cell density. These changes of membrane calcium uptake activity occur promptly and do not require protein synthesis.     

Thyroid Hormone Binding and Regulation of Adrenergic Enzymes in Two Neuroblastoma Cell Lines

Two neuroblastoma cell lines were cultured in control (euthyroid) and hypothyroid media and examined for protein, RNA and DNA content, activity of the catecholaminergic enzymes tyrosine hydroxylase (TH, EC 1.14.16.2) and monoamine oxidase-A (MAO-A, EC 1.4.3.4), and for L-triiodothyronine (T3) nuclear receptors. In the hypothyroid condition, the rate of cell division and the levels of RNA and protein as well as the activities of TH and MAO were lower than in the euthyroid condition, the reduction being more marked in the E than in the A2(1) cell line. T3 nuclear receptors, unaltered in affinity, were increased in number in the hypothyroid medium, possibly as a regulatory response to hormonal deficiency. Examination of a possible relationship between T3 occupancy and TH activity in the E cells, most sensitive to thyroid hormone deficiency, revealed that induction of TH activity by T3 is dose-dependent and correlates with the number of nuclear sites occupied by the hormone. When neuroblastoma cells were induced to differentiate by the addition of sodium butyrate to the medium, parameters of cell growth (protein, RNA) and enzyme activity (TH and MAO-A) increased in both cell lines irrespective of the presence of thyroid hormones. These data indicate that thyroid hormones, through their nuclear receptors, directly affect the activity of catecholaminergic enzymes in cultured, immature (undifferentiated) neurons.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

Effects of Electromagnetic Fields on the Bioactivities of an Osteoblast-Like Cell Line (UMR-106)

In the present study, we first investigated the effects of various types of low-energy, low-frequency electromagnetic fields (EMFs) on DNA synthesis in UMR-106 osteoblast-like cells. The experimental groups were exposed to EMFs for 2 days (twice/day, 30 min/time), and DNA synthesis was measured. The results showed that the cells responded most sensitively to EMFs of some specific combinations of the parameters by an increase in DNA synthesis, implying that EMFs with a specific waveform rather than a complex one can be used in clinical electrotherapy. The parameters were as follows: pulsed electric field (PEF) with pulse width 0.2 ms, field strength 10 V/cm, frequency 125 Hz; sinusoidal electric field (SEF) with field strength 1 V/cm, frequency 10 Hz; and alternating magnetic field (AMF) with field density 0.5 mT, frequency 5 Hz. In addition to frequency, the field strength or field density within a suitable intensity scale played a dominant role in causing the DNA synthesis response. We then compared the effects of two kinds of fields, PEF and AMF, with the optimum parameters identified by the experiments, on alkaline phosphatase (ALP) activity, protein and collagen synthesis, and intracellular levels of cyclic adenosine monophosphate (cAMP). The results indicated that both fields could not only affect UMR-106 cells proliferation but could particularly affect a series of characteristic bioactivities of UMR-106 such as ALP activity and collagen synthesis. The intracellular cAMP levels were increased rapidly and greatly with exposure to both PEF and AMF, implying that the action of low-frequency EMFs proceeds via second messenger-dependent processes originating from signals at the cell membrane. The difference in action between PEF and AMF suggests that they may couple to the cell membrane in a partially different way.     

A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid

A new strain of Butyrivibrio fibrisolvens (TH1) that has high potential to produce conjugated linoleic acid (CLA) was isolated. Strain TH1 had higher LA isomerase (LA-I) activity, and was much more tolerant to linoleic acid (LA) than other strains examined. However, high CLA reductase (CLA-R) activity resulted in the temporary accumulation of CLA and subsequent conversion to trans-vaccenic acid (t-VA). When LA was added to growing TH1 cultures in a solution with dimethylsulfoxide (LA/DMSO), CLA produced was greater than when LA was added in a mixture with bovine serum albumin (BSA). The number of viable cells decreased upon addition of LA/DMSO, but then increased as the CLA decreased upon its conversion to t-VA. This result suggests that B. fibrisolvens can resume growing by the removal of CLA from the cells. Most CLA was released from B. fibrisolvens cells by gentle washing with BSA, suggesting that CLA bound to the cells might be removed in the rumen and large intestine. Thus, CLA production by B. fibrisolvens in the digestive tract could be increased by a reduction in CLA-R activity without accompanying an overall decrease in the cell number of B. fibrisolvens. Fatty acids (FAs) with 18 carbon backbone inducted LA-I activity, whereas unsaturated FAs induced CLA-R activity, suggesting that FAs stimulate the synthesis of LA-I and CLA-R. Providing a diet with a low ratio of unsaturated to saturated FAs may favor CLA production.     

Deoxyribonuclease II activity in relation to cell cycle in synchronized HeLa S3 cells

Studying the activity of DNase II in relation to cell cycle in synchronized HeLa S3 cells show a two to seven fold increase in DNase II activity at those times when DNA synthesis is taking place. The peaks of DNase II activity coincide with the peaks of DNA synthesis. The increased DNase II activity could be prevented by puromycin, suggesting that the enzyme activity increased at the S phase was caused by synthesis of new molecules rather than the activation of existing molecules. Acid phosphatase (as a marker for lysosomal enzymes) does not show an induction similar to that observed for DNase II in relation to cell cycle.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.