Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine

We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells. Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes. Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity. Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values. Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value. After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen. In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250%. 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled. The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol. In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity. The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol. On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures. It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.     

Independent inhibition of DNA synthesis by protein kinase C, cyclic AMP and interferon alpha/beta in rabbit aortic smooth muscle cells

In quiescent cultures of rabbit aortic smooth muscle cells, whole blood serum-induced DNA synthesis was inhibited markedly by protein kinase C-activating 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12, 13-dibutyrate (PDBu), cyclic AMP-derivatives, such as dibutyryl cyclic AMP (Bt2cAMP) and 8-bromo-cyclic AMP, and interferon alpha/beta. Neither TPA nor interferon alpha/beta elevated the cellular cyclic AMP level. Neither Bt2cAMP nor interferon alpha/beta induced the phospholipase C-mediated hydrolysis of phosphoinositides. The down-regulation of protein kinase C by prolonged treatment with PDBu abolished the antiproliferative action of TPA but did not affect that of Bt2cAMP or interferon alpha/beta. TPA and Bt2cAMP inhibited the serum-induced DNA synthesis when added within 12 h after the addition of the serum, while interferon alpha/beta was active only when added within 6 h. These results suggest that there are at least three independent signaling systems, protein kinase C- and cyclic AMP-mediated systems and an unidentified system for interferon alpha/beta, which are involved in the antiproliferative mechanisms in rabbit aortic smooth muscle cells.     

cAMP is an essential signal in the induction of antibody production by B cells but inhibits helper function of T cells

Dibutyryl cAMP and IL 1 were found to stimulate antigen-specific and polyclonal antibody production when added together to cultures of highly purified B cells. We propose that IL 1 and an elevation in cytoplasmic cAMP represent minimal signal requirements for B cell activation. In contrast to its effect on B cells, dibutyryl cAMP inhibited helper T cell activity. Cyclic AMP suppressed the production of IL 2 and T cell replacing factor (TRF) by T cells and thus abrogated the ability of helper T cells to enhance SRBC-specific antibody production by B cells. Cyclic AMP did not inhibit the generation by T cells of B cell growth factor (BCGF). BCGF, not normally detected in Con A supernatant, was found in the culture supernatant of spleen cells that were stimulated with Con A in the presence of cAMP. Our findings indicate that cAMP blocks the production of an inhibitor of BCGF activity. cAMP had no effect on the production by macrophages of IL 1.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.     

Induction of alkaline phosphatase in choriocarcinoma cells by 1-beta-D-arabinofuranosyl-cytosine, mitomycin C, phleomycin, and cyclic nucleotides.

Alkaline phosphatase is induced in cultured human choriocarcinoma cells by three inhibitors of DNA synthesis which alter DNA structure: 1-β-D-arabinofuranosyl-cytosine, mitomycin C, and phleomycin. No induction is observed with the inhibitors, hydroxyurea and thymidine, which do not alter DNA structure. Cyclic AMP, analogs of cyclic nucleotides, and sodium butyrate also induce alkaline phosphatase in these cells. Among the cyclic nucleotides tested, dibutyryl cyclic AMP is the best inducer, whereas dibutyryl cyclic GMP is a poor inducer. Induction of alkaline phosphatase by inhibitors of DNA synthesis or by exposure to dibutyryl cyclic AMP appears to utilize different mechanisms. Maximum induction is observed after simultaneous addition of both types of inducers at the concentrations found to be optimal for each inducer alone. Under these conditions, the induced activity is equal to or greater than the sum of the activities induced by each inducer. RNA synthesis and protein synthesis are required for induction. Dibutyryl cyclic AMP added to cultures of choriocarcinoma cells is not degraded in the culture medium, but is extensively degraded in the cells. Nevertheless, significant amounts of dibutyryl and monobutyryl cyclic AMP are found intracellularly throughout the experiment. Since the cellular uptake of dibutyryl cyclic AMP is extremely slow, the amount of butyrate released by intracellular degradation cannot account for the observed induction. Neither the rate of uptake nor the stability of dibutyryl cyclic AMP are changed by the addition of 1-β-D-arabinofuranosyl-cytosine to the culture medium. Furthermore, 1-β-D-arabinofuranosyl-cytosine inhibits the induction by sodium butyrate. The results indicate that butyrate is not the major mediator of induction by dibutyryl cyclic AMP.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

Hormonal regulation of fatty acid synthetase in chick embryo liver.

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.     

Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin-1

When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process-bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half-maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin-1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300-1,000-fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase-1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP-induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.     

Interleukin 2 regulates immune interferon (IFN gamma) production by normal and suppressor cell cultures

A Lyt-2+ lymphocyte is responsible for immune interferon (IFN gamma) production in mitogen-stimulated mouse spleen cell cultures. A Lyt-1+ helper cell is required for the induction of IFN gamma. Interleukin 2 (IL 2) can specifically replace the Lyt 1 cell requirement. IL 2 will also abrogate suppressor cell inhibition of IFN gamma production. IFN gamma production appears to be regulated by a dynamic interaction between helper cells (source of IL 2), suppressor cells (absorption of IL 2?) and IFN gammna-producer cells.     

Regulation of ornithine decarboxylase activity by amino acids, cyclic AMP and luteinizing hormone in cultured mammalian cells

Any one of five amino acis (alanine, asparagine, glutamine, glycine, and serine) is an essential requirement for the induction of ornithine decarboxylase (EC 4.1.1.17) in cultured chinese hamster ovary (CHO) cells maintained with a salts/glucose, medium. Each of these amino acids induced a striking activation of ornithine decarboxylase in the presence of dibutyryl cyclic AMP and luteinizing hormone. The effect of the other amino acids was considerably less or negligible. The active amino acids at optimal concentrations (10 mM) induced only a 10-20 fold enhancement of enzyme activity alone, while in the presence of dibutyryl cyclic AMP, ornithine decarboxylase activity was increased 40-50 fold within 7-8 h. Of the hormones and drugs tested, luteinizing hormone resulted in the highest (300-500 fold) induction of ornithine decarboxylase with optimal concentrations of dibutyryl cyclic AMP and asparagnine. Omission of dibutyryl cyclic AMP reduced this maximal activation to one half while optimal levels of luteinizing hormone alone caused no enhancement of ornithine decarboxylase activity. The induction of ornithine decarboxylase elicited by dibutyryl cyclic AMP, amino acid and luteinizing hormone was diminished about 50% with inhibitors of RNA and protein synthesis. The specific amino acid requirements for ornithine decarboxylase induction in chinese hamster ovary cells was similar to the requirements for induction in two other transformed cell lines. Understanding the mechanism of enzyme induction requires an identification of the essential components of the regulatory system. The essential requirement for enzyme induction is one of five amino acids. The induction of ornithine decarboxylase by dibutyryl cyclic AMP and luteinizing hormone was additive in the presence of an active amino acid.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

Differential Expression of Type A and Type B Monoamine Oxidase of Mouse Astrocytes in Primary Cultures

Abstract: Type A and type B monoamine oxidase (MAO) activities were determined in mouse brain and in primary cultures of mouse astrocytes. Thirty-one-day-old astrocyte cultures exhibited predominantly type A MAO activity. In cultures of the same age, treated with 0.25 mM dibutyryl cyclic AMP under the same culturing conditions, 30% type B MAO was expressed, although dibutyryl cyclic AMP up to 1 mM does not affect MAO activity in vitro. The specific activity of type B MAO increased significantly in older cultures, while type A MAO changed only slightly.     

Changes in enzyme patterns produced by high potassium concentration and dibutyryl cyclic AMP in organ cultures of sympathetic ganglia

—Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P < 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide. In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [³H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [³H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP. The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm -potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP. Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.     

Effects of granulocyte-macrophage colony-stimulating factor and cyclic AMP interaction on human neutrophil apoptosis.

The current study was undertaken to evaluate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclic AMP (cAMP) signaling interaction on human neutrophil apoptosis, either occurring spontaneously or induced by Fas antigen activation. Results show that GM-CSF, dibutyryl cAMP (a cAMP analog) and forskolin (an adenylate cyclase activator) are all able to suppress spontaneous neutrophil cell death. Of note however, when GM-CSF is used in combination with cAMP-elevating agents, an additive effect on neutrophil survival is observed with dibutyryl cAMP only, whereas supplementation of cell cultures with GM-CSF and forskolin results in a progressive reduction of antiapoptotic effects exerted by the single compounds. Moreover, although dibutyryl cAMP and forskolin do not affect Fas-triggered apoptotic events, they are still able to modulate the GM-CSF capacity to prolong neutrophil survival following anti-Fas IgM cell challenge, with effects similar to those respectively exerted on spontaneous neutrophil apoptosis. The data indicate that GM-CSF may negatively modulate the cAMP-mediated antiapoptotic pathway in human neutrophils, likely via the inhibition of adenylate cyclase activity. This would prevent an abnormal neutrophil survival as a result of cAMP signaling stimulation, which provides a novel insight into the role of GM-CSF as a physiological regulator of myeloid cell turnover.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.