Calcium ions as a factor of cell-cell cooperation in hepatocyte cultures

Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.     

Age-related features of protein synthesis rhythm in hepatocytes. Effects of extracellular medium

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and alpha1-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.     

Loss of hepatocyte co-operative activity after inhibition of ganglioside GM1 synthesis and shedding

Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity.     

Age-related features of protein synthesis rhythm. Effects of extracellular medium

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and ₁-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 9–17.Original Russian Text Copyright © 2005 by Brodsky, Nechaeva, Zvezdina, Novikova, Gvazava, Fateeva, Malchenko.     

Involvement of protein kinases in self-organization of the rhythm of protein synthesis by direct cell-cell communication

Primary cultures of rat hepatocytes grown on slides were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of synchronization of individual oscillations, resulting in the formation of a common rhythm of the cell population, i.e. cell-cell self-organization. Dense synchronous and sparse non-synchronous cultures were used to estimate effect of protein kinase activity on the kinetics of protein synthesis. Treatment of dense cultures with the inhibitors H7 (40 microM) or H8 (25 microM) resulted in a loss of the protein synthesis rhythm, a suppression of the cell-cell self-organization. Stimulation of protein kinase activity with either 0.5 or 1.0 microM phorbol 12-miristate-13-acetate (PMA) or 10 microM forskolin caused the appearance of the synthetic rhythm in non-synchronous sparse cultures under otherwise normal conditions. Inhibition of protein kinase activity with H7 resulted in signal factors, such as gangliosides and phenylephrine, failing to initiate this rhythm in sparse cultures. Activation of protein kinase activity with PMA shifted the phase pattern of the protein synthesis rhythm. Thus, according to our previous and the new data, protein kinase activity and consequently protein phosphorylation is the crucial step of sequence of processes resulting in synchronization during self-organization of cells in producing a common rhythm in the population. The general pathway can be presented as follows: signaling of gangliosides or other calcium agonists-->efflux of calcium ion from intracellular stores, with elevation of calcium concentration in the cytoplasm-->activation of protein kinases-->protein phosphorylation-->synchronization of individual oscillations in protein synthesis rates-->induction of a common rhythm throughout this population. The data have been discussed concerning similarity of the direct cell-cell communication and the cell self-organization in cultures and in organism.     

Glutamic acid as enhancer of protein synthesis kinetics in hepatocytes from old rats

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell–cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell–cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.     

Melatonin promotes and synchronizes protein synthesis in hepatocyte culture from old rats

The effect of 1 to 1000 nM melatonin was studied on daily cultures of rat hepatocytes on slides in serum-free medium. The minimum melatonin concentration (1 nM) proved to synchronize protein synthesis in asynchronous sparse cultures of hepatocytes from rats of different age, and a circahoralian rhythm of protein synthesis was revealed in them. In dense weekly synchronous hepatocytes from old rats (2.5 years old with the weight of about 600 g), melatonin improved cell synchronization to the level of young animals. Melatonin treatment increased the mean rate of protein synthesis in rats of different age.     

Glutamic acid – amino acid,neurotransmitter, and drug – is responsible for protein synthesis rhythm in hepatocyte populations <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell–cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.     

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.     

Self-synchronization of the protein synthesis rhythm in HaCaT cultures of human keratinocytes

In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of protein synthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of ³H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.     

Glutamic Acid Signal Synchronizes Protein Synthesis Kinetics in Hepatocytes from Old Rats for the Following Several Days. Cell Metabolism Memory

The kinetics of protein synthesis was investigated in primary cultures of hepatocytes from old rats in serum–free medium. The rats were fed mixed fodder supplemented with glutamic acid and then transferred to a regular mixed fodder. The amplitude of protein synthesis rhythm in hepatocytes isolated from these rats increased on average 2–fold in comparison with the rats not receiving glutamic acid supplement. Based on this indicator reflecting the degree of cell–cell interactions, the cells from old rats were not different from those of young rats. The effect was preserved for 3–4 days. These results are discussed in connection with our previous data on preservation of the effect of single administration of gangliosides, noradrenaline, serotonin, and other synchronizers on various cell populations. In contrast to the other investigated factors, glutamic acid is capable of penetrating the blood–brain barrier, which makes its effect possible not only in the case of hepatocytes and other non–brain cells, but also in neurons.     

Ganglioside-mediated metabolic synchronization of protein synthesis activity in cultured hepatocytes

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.     

Melatonin as the most effective organizer of the rhythm of protein synthesis in hepatocytes in vitro and in vivo

Recent data has extended a large array of melatonin functions by the discovery of melatonin's involvement in the organization and regulation of the rhythm of intracellular protein synthesis. An ultradian rhythm in total protein synthesis has been detected in primary hepatocyte cultures 5 min after addition of 1-5 nM melatonin to the medium. The melatonin effect was mediated via its receptors (as shown in experiments with luzindole), leading to the cell synchronization as well as the mean rate of protein synthesis rate being increased. The chain of processes synchronizing the oscillation of the rate protein synthesis throughout the hepatocyte population includes Ca2+ fluxes {experiments with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetomethyl ester)]}. Inhibition of protein kinase activity (experiments with H7) inhibited the synchronizing function of melatonin. Activation of protein kinase activity results in a shift of the protein synthesis oscillation; the effect was the same as melatonin added to the culture medium. In another series of experiments, after melatonin was intraperitoneally injected to rat (0.015-0.020 μg/kg), hepatocytes were isolated and cultures established. A synchronizing effect of melatonin in vivo was detected as early as in the estimates from the direct action of melatonin on cell cultures. In the cultures obtained from old rats provided with melatonin, the amplitude of protein synthesis rhythm was enhanced, i.e. cell-cell interactions were increased, as well as rate of the protein synthesis being enhanced.     

Single short-term signal that enhances cooperative activity of old rat hepatocytes acts for several days

Primary cultures of rat hepatocytes were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of overall cell synchronization and cooperation amongst the population. The level of synchronization was determined by amplitudes of the rhythm. Low synchronization of old rat hepatocytes can be enhanced by addition of either gangliosides or phenylephrine to the medium. Incubation of cultures with gangliosides lasted for 2.5 h, while action of phenylephrine was only for 2 min. The amplitude of protein synthesis rhythm was increased 1.5-2 times. In cultures transferred to a fresh normal medium, this increased amplitude was observed for at least 2-3 days. Thus, both gangliosides and phenyleprine are triggers, which, as shown earlier, initiated calcium-dependent processes in the cytoplasm. The results are discussed in the light of concept of the cell self-organization by a direct cell-cell communication.     

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 μM or forskolin at 10 μM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 μM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 μM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists → changes in the level of calcium ions in cytoplasm → increased protein kinase activities → protein phosphorylation → modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.     

Density dependent proliferation of human glia cells stimulated by epidermal growth factor.

Epidermal growth factor (EGF) isolated from mouse salivary glands, enhanced the multiplication and [³H]TdR incorporation of human normal glia cells in serum-free medium supplemented with human serum albumin. Optimal dose was 2 ng/ml for both dense and sparse cultures but dense cultures were stimulated by EGF to a much less extent than sparse cultures. Data are presented that make the possibility unlikely that the density dependent inhibition of the EGF response is due to depletion of EGF in the medium or a local, juxtacellular starvation for the factor.     

Disturbed Cooperation of Hepatocytes in the Rhythm of Protein Synthesis by Chelating Agent of Cytoplasmic Calcium BAPTA-AM

Previously we demonstrated synchronized oscillations of protein synthesis rate in hepatocyte cultures upon accumulation of monosialoganglioside GM1 in the medium or after introduction of exogenous GM1 to the medium. The synchronized oscillations of the protein synthesis rate in dense hepatocyte cultures were blocked 30 min after their treatment with 10–20 M BAPTA-AM, a chelating agent of cytoplasmic calcium. Enzyme immunoassay for GM1 demonstrated similar amounts of GM1 in the medium conditioned for 3 h by dense hepatocyte cultures pretreated with 20 M BAPTA-AM for 1 h and in the medium of normal dense cultures: 0.0060 ± 0.0005 and 0.0055 ± 0.0005 pmol/1000 cells, respectively. The content of GM1 was also similar in the normal and BAPTA-AM-pretreated hepatocytes: 0.158 ± 0.013 and 0.183 ± 0.014 pmol/1000 cells, respectively. The synchronized rhythm of protein synthesis has been confirmed in the diluted cultures in the medium conditioned by the normal dense cultures. However, the medium conditioned by the dense cultures pretreated with BAPTA-AM induced no synchronization of the diluted cultures. Since GM1 concentration was normal in this medium, we propose the effect of a physicochemical form of the gangliosides accumulated in the medium on their ability to synchronize the rhythm of protein synthesis.     

The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells

Addition of 0.14 microM dexamethasone (DEX) to young log-phase WI38 cultures seeded at various densities in serum-free medium containing 4.1 nM epidermal growth factor (EGF) resulted in a synergistic increase in proliferation and final cell density. The action of DEX plus EGF was stimulatory but not synergistic in young confluent cultures. DEX plus EGF had no synergistic effect on senescent cells either during log phase or at confluence. Analysis of the effect of DEX on [125I]EGF binding revealed no statistically significant changes in either the number of binding sites or the apparent dissociation constant of the EGF-receptor complex.     

Cooperation of Hepatocytes in vitro in the Rhythm of Protein Synthesis is Promoted by the GM1 Ganglioside Contained in Vesicles and Liposomes

The medium conditioned by dense, self-synchronized hepatocyte cultures was centrifuged at 150000 g to obtain two fractions. The light fraction (supernatant fluid) contained ganglioside monomers and micelles, and the heavy fraction (pellet) contained gangliosides in the vesicles shed from the cell membrane. In the test populations of hepatocytes, the rhythm of protein synthesis was used as an indicator of cell synchronization resulting from their cooperative activity. Diluted hepatocyte cultures with asynchronous fluctuations of protein synthesis proved to be synchronized by both the initial conditioned medium and its vesicular fraction. Our previous studies have shown that this occurs under the effect of GM1 monosialoganglioside, which is released from cultured cells and accumulated in the conditioned medium. Liposomes consisting of GM1 and phosphatidylcholine from egg yolk (1 : 19 mol%), compared to free exogenous GM1, synchronized the rhythm of protein synthesis more effectively: synchronization was observed at a GM1 concentration in liposome suspension of only 0.0003 M, compared to 0.06 M and higher in the case of free GM1. Thus, GM1 as a component of membranes and monolayer lipid structures proved to be much more effective than free GM1 in promoting hepatocyte cooperation with respect to the rhythm of protein synthesis.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.