Zinc transferrin. Enhancement of nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

Zinc transferrin, when added to serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, causes an increase in deoxyribonucleic acid synthesis over that seen with phytohemagglutinin alone, as judged by the uptake of tritiated thymidine. This effect is not seen with zinc acetate, zinc albumin, or zinc ovotransferrin. Zinc transferrin also has a similar effect on ribonucleic acid synthesis. Furthermore, transferrin-bound zinc is specifically taken up by stimulated lymphocytes, maximal uptake occurring approximately 14 hr after the addition of phytohemagglutinin. These results indicate a function for serum transferrin in zinc metabolism, and, moreover, a role for the zinc transferrin complex in lymphocyte metabolism.     

Effect of iron transferrin on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

In serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, iron transferrin causes enhanced uptake of both tritiated thymidine and tritiated uridine over that seen with only phytohemagglutinin. This effect is specific for the iron transferrin complex, no enhancement produced by either free iron(III) or apotransferrin. Iron bound to transferrin is quantitatively taken up by stimulated lymphocyte cultures, while under similar conditions only 10% of transferrin-bound zinc is incorporated. The relative specificity of action of iron and zinc on nucleic acid synthesis is discussed.     

Molecular distribution of cytoplasmic zinc in phytohemagglutinin-stimulated and unstimulated human lymphocytes

Human peripheral blood lymphocytes were incubated with [⁶⁵Zn] zinc transferrin and with and without phytohemagglutinin for 1, 2, 4, 10, 24, 48, 72, and 96h. Gel filtration of cytoplasmic fractions obtained from these lymphocytes was then performed to determine the molecular distribution of incorporated zinc as a function of time in culture. The data obtained indicated that: (1) transferrin-bound zinc incorporated by human lymphocytes is associated with a variety of soluble molecules whose molecular weights range from less than 5,000 to greater than 70,000 daltons; (2) there is a time-dependent change in the distribution of cytoplasmic zinc for both phytohemagglutinin-stimulated and unstimulated lymphocytes; and (3) for all times studied, there is a difference in the elution profiles obtained for phytohemagglutinin-stimulated and unstimulated lymphocytes. Furthermore, lymphocytes from a donor with untreated hairy cell leukemia exhibited a totally different pattern of cytoplasmic zinc distribution than did lymphocytes from apparently healthy donors.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Studies on the cyclic AMP response to prostaglandin in human lymphocytes

It is generally thought that cyclic AMP acts as the second messenger for prostaglandin E in human lymphocytes. We have recently found that the mitogen-induced proliferation of human lymphocytes is no longer inhibited by PGE₂ if the lymphocytes are preincubated overnight prior to the addition of mitogens and PGE₂. In this paper we report that lymphocytes also lose their cyclic AMP response to mitogens after preincubation. The loss of sensitivity to PGE with preincubation can be blocked by cyclohexamide (25 μg/ml). Indomethacin (1 μg/ml) partially blocked the loss of sensitivity, but removal of the glass-adherent cells did not. Since either manipulation effectively stops prostaglandin production in the preincubation cultures, it would appear that indomethacin prevented the loss of sensitivity to PGE₂ by a mechanism other than inhibition of PG synthetase. The addition of phytohemagglutinin to the preincubation cultures also blocked the loss of sensitivity to PGE₂.     

In vitro immune response of human peripheral lymphocytes. III. Effect of anti-mu or anti-delta antibody on PWM-induced increase of cyclic nucleotides in human B lymphocytes.

Stimulation of human peripheral blood lymphocytes (PBL) with pokeweed mitogen (PWM) induced consistent increases of intracellular levels of cyclic AMP and cyclic GMP within 15 min. Increases of cyclic AMP were observed in both B and T lymphocyte populations, but increase of cyclic GMP was observed only in the B lymphocyte population. The addition of anti-mu antibody to B cells abolished PWM-induced increase of cyclic GMP without any effect on cyclic AMP response. Anti-delta antibody did not show any inhibitory or stimulatory effect on PWM-induced increase of cyclic GMP or cyclic AMP. Pretreatment of B cells with anti-mu antibody at 37 degrees C for 1 hr inhibited PWM-induced increase of cyclic GMP, whereas pretreatment with anti-mu antibody at 4 degrees C did not show any inhibitory effect on PWM-induced increase of cyclic GMP. The effect of anti-mu-pretreatment was reversible and pretreated cells were recovered from the inhibitory effect of anti-mu antibody after 36 hr culture.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.     

Activation of murine lymphocytes by cyclic guanosine 3′,5′-monophosphate: Specificity and role in mitogen action

Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) stimulates nucleic acid synthesis in lymphocytes, and has been implicated as the intracellular effector of the actions of mitogenic agents on these cells. In the present study, we examined the specificity of the mitogenic activity of cyclic GMP and of its 8-bromo (Br) derivatives, and the effects of the T cell mitogens, concanavalin A, phytohemagglutinin, and staphylococcal entertoxin B (SEB) on the cyclic GMP content and guanylate cyclase activity of mouse splenic lymphocytes. Cyclic GMP and guanosine modestly increased the incorporation of [³H]thymidine into DNA by cultured lymphocytes, but were far less effective than their 8-Br-derivatives. However, on a molar basis the mitogenic activity of both 8-Br-guanosine and 8-Br-5′-GMP exceeded that of 8-Br-cyclic GMP, when tested in the presence and absence of serum in the culture media. Combined addition of maximal doses of these nucleotides did not give additive stimulatory effects, suggesting an action on a common subpopulation of cells, and possibly a common mechanism. By contrast, cyclic AMP, 8-Br-cyclic AMP, 8-Br-adenosine, cholera toxin and prostaglandin E₁ suppressed both basal [³]thymidine incorporation and stimulation of this parameter by T-cell line mitogens and the guanosine nucleotides. Rapid effects of concanavalin A, phytohemagglutinin, SEB, guanosine, 5′-GMP, 8-Br-guanosine, and 8-Br-5′-GMP on the cyclic GMP content of murine lymphocytes could not be demonstrated. Similarly, concanalin A, phytohemagglutinin and SEB failed to alter guanylate cyclase activity when added directly to cellular homogenates or pre-incubated with intact cels. Conversely, carbamylcholine rapidly increased lymphocyte cyclic GMP but was not mitogenic.These results are consistent with the hypothesis that cyclic GMP and cyclic AMP are antagonistic in their influence on lymphocyte mitogenesis. However, they also demonstrate that related nucleotides are more potent mitogens than cyclic GMP and suggest that activation of murine lymphocytes by concanavalin A, phytohemagglutinin and SEB may not be mediated by rapid increases in cellular cyclic GMP content. Since high concentrations of exogenous cyclic GMP and related nucleotides must be used to influence DNA synthesis, the biologic significance of this effect remains uncertain.     

Modulation of peripheral blood mononuclear cell cyclic adenosine monophosphate levels by human very low density lipoprotein

Normal human plasma very low density lipoprotein (VLDL) can inhibit mitogen-induced lymphocyte DNA synthesis. Since early events in lymphocyte activation (e.g., cyclic nucleotide metabolism) are thought to influence the magnitude of later events (e.g., [³H]thymidine uptake) we designed the current studies to compare the effects of VLDL on these two cellular processes. Two separate effects of VLDL on peripheral blood mononuclear cell (PBM) cyclic adenosine monophosphate (AMP) metabolism were observed at VLDL concentrations which inhibit phytohemagglutinin (PHA)-induced [³H]thymidine uptake. VLDL suppressed the early, transient increase in PBM cyclic AMP which occurs within minutes of the addition of mitogen. VLDL exposure also stimulated a delayed (greater than 24 hr) and spontaneous increase in PBM cyclic AMP levels which corresponded temporally with progressive cellular refractoriness to mitogen stimulation. If mitogen-induced lymphoproliferation is influenced by early changes in cyclic nucleotide metabolism, as claimed by some investigators, then perhaps the ability of VLDL to modulate intracellular cyclic AMP levels may explain some of the antiproliferative properties of this bioregulatory lipoprotein.     

Improved methods for obtaining colonies of human peripheral blood lymphocytes in vitro for radiation dose-survival studies

Culture conditions were examined for colony formation of human peripheral blood lymphocytes in vitro. It was found that addition of 1% human serum to the medium together with fetal calf serum greatly improved the cloning efficiency and colony size, moreover allogeneic lymphocytes and lymphoblastoid cells are both required as feeder cells for better results. The X-ray dose-survival study showed that the radiosensitivity of lymphocytes remained essentially the same whether the irradiation was performed prior to separation of the lymphocytes from blood or 4 h after addition of phytohemagglutinin to the separated lymphocyte culture; however, the sensitivity was definitely increased as the cell cycle progressed from G₀ to G₁/S or log phase.     

Subcellular distribution of transferrin-bound zinc incorporated by phytohemagglutinin-stimulated and unstimulated human lymphocytes

The distribution of transferrin-bound zinc incorporated by phytohemagglutinin-stimulated and unstimulated human lymphocytes has been studied as a function of time in four subcellular fractions (nuclei, mitochondria, microsomes, and soluble). In untreated lymphocytes, the percent of total incorporated zinc in each fraction remains relatively constant over 72 h in culture. However, there is a time-dependent change in the percent of total incorporated zinc in all fractions isolated from phytohemagglutinin-stimulated cells, and this change is most apparent for the nuclear and soluble fractions. Apparently some sustained production of energy is required for this change in subcellular distribution of zinc to occur. Additionally, the uptake of cytoplasmic zinc by purified lymphocyte nuclei has been studied. Uptake is rapid and occurs maximally under conditions known to be optimal for stimulation of nuclear adenylate cyclase.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Pyridine nucleotide coenzyme levels in phytohemagglutinin-stimulated human lymphocytes

A radioisotopic, enzymatic cycling procedure was used to measure NAD, NADH, NADP and NADPH in cultured human lymphocytes at 0, 24 and 48 h after exposure to phytohemagglutinin (PHA). During the 0–24 h period after PHA addition NAD and NADH were increased in both control and test cultures leading to a decrease in the NAD: NADH ratio. During the 24–48 h period increases in NAD and NADH occurred in test cultures in parallel with increased incorporation of [³H]TdR. No change in the NAD: NADH ratio was seen. The results indicate that the levels of NAD and NADH may be affected by the culture conditions and that increases in these compounds occur in stimulated cells during a time period in which DNA turnover is elevated and cell volume is increased but before extensive cell division.     

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN₃, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl₂, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the sameThus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.     

The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: relationship of enzyme activity to cyclic AMP concentrations.

The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX greater than theophylline greater than caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2'-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cycli AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

The mechanism by which 2-deoxyglucose inhibits glucose-induced activation of Pilobolus longipes spores

Sporangiospores of Pilobolus longipes were activated by either glucose, 6-deoxyglucose, or derivatives of cyclic AMP. Cyclic AMP content increased after the addition of either glucose or 6-deoxyglucose and the increase preceded spore activation, indicating that glucose triggers germination via cyclic AMP. Activation, whether induced by glucose, 6-deoxyglucose, or cyclic nucleotides was inhibited by 2-deoxyglucose. However, cyclic AMP levels also increased after the addition of 2-deoxyglucose. Radioactive 2-deoxyglucose was recovered from spores mainly as 2-deoxyglucose 6-phosphate, suggesting that phosphorylation of 2-deoxyglucose may inhibit spore activation by trapping ATP. Support for the hypothesis came from ATP assays which showed that 2-deoxyglucose reduced intracellular ATP to undetectable levels. Moreover, when ATP levels were restored with exogenous fructose, 2-deoxyglucose was no longer inhibitory but was then an effective germination trigger.     

Cyclic AMP-Mediated Enhancement of High-Affinity Choline Transport and Acetylcholine Synthesis in Brain

Abstract: Intracerebroventricular administration of N⁶, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time-dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent V_max without a change of the K_m for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.