Lead inhibition of enzyme synthesis in soil.

Addition of 2 mg of Pb2+/g of soil concident with or after amendment with starch or maltose resulted in 75 and 50% decreases in net synthesis of amylase and alpha-glucosidase, respectively. Invertase synthesis in sucrose-amended soil was transiently reduced after Pb2+ addition. Amylase activity was several times less sensitive to Pb2+ inhibition than was enzyme synthesis. In most cases, the rate of enzyme synthesis returned to control (Pb2+) values 24 to 48 h after the addition of Pb. The decrease in amylase synthesis was paralleled by a decrease in the number of Pb-sensitive, amylase-producing bacteria, whereas recovery of synthesis was associated with an increase in the number of amylase-producing bacteria. The degree of inhibition of enzyme synthesis was related to the quantity of Pb added and to the specific form of lead. PbSO4 decreased amylase synthesis at concentrations of 10.2 mg of Pb2+/g of soil or more, whereas PbO did not inhibit amylase synthesis at 13 mg of Pb2+/g of soil. Lead acetate, PbCl2, and PbS reduced amylase synthesis at total Pb2+ concentrations of 0.45 mg of Pb2+/g of soil or higher. The results indicated that lead is a potent but somewhat selective inhibitor of enzyme synthesis in soil, and that highly insoluble lead compounds, such as PbS, may be potent modifiers of soil biological activity.     

Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis

The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3' poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.     

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.     

Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.     

Synthesis of sterols and 5-lipoxygenase products are required for the G1-S phase transition of interleukin-2-dependent lymphocyte proliferation

A murine killer T cell line, G-CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL-2), was used to analyze the mechanism of IL-2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL-2. Compactin, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate-limiting step of the sterol synthesis), inhibited the IL-2-induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL-2 action. The IL-2-induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5-lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5-lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5-lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL-2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.     

Obligatory action of polypeptide growth factors for the IL-1-mediated prostaglandin E2 production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-1

Our studies show that in connective tissue cells, induction of PGE2 synthesis in response to IL-1 requires costimulation with platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). In cells incubated in medium containing fresh serum, IL-1 induced a dose-dependent synthesis of PGE2. However, when the cells were incubated in medium containing low serum or platelet poor plasma (lacking PDGF), IL-1 alone failed to induce PGE2 synthesis. PGE2 synthesis was restored when platelet poor plasma was supplemented with PDGF. Addition of PDGF or FGF together with IL-1 resulted in a 14- and 66-fold stimulation of PGE2 synthesis, respectively. Stimulation was dependent on the concentration of both IL-1 and the growth factor. PGE2 synthesis was also dependent on the synthesis of new proteins. In cells simultaneously treated with IL-1 and PDGF, PGE2 synthesis was initiated after a lag of 2 to 3 h, proceeded first with a rapid rate for 6 h, and then with a slower rate through 24 h. PGE2 synthesis during the latter, slower phase was greatly enhanced by pretreatment with PDGF, but not by pretreatment with IL-1. PDGF pretreatment also resulted in maintenance of 10- to 12-fold higher cell surface IL-1-binding during this phase. These data provide evidence for potentially novel interactions between PDGF and IL-1 activities, one of which is the modulation of IL-1 receptors by PDGF. Furthermore, these studies suggest that by virtue of their effect on IL-1 activities, PDGF and FGF may play additional roles in connective tissues, including an indirect role in inflammatory processes.     

Presynaptic Adenosine A2 and N-Methyl-D-Aspartate Receptors Regulate Dopamine Synthesis in Rat Striatal Synaptosomes

Dopamine synthesis rate and cyclic AMP concentration were measured in synaptosomes prepared from rat striatum. Dopamine synthesis rate was decreased by the addition of either adenosine deaminase or 8-phenyltheophylline, an adenosine receptor blocker, and was increased by the addition of 2-chloroadenosine. The addition of L-glutamate in the absence of adenosine deaminase decreased both dopamine synthesis rate and cyclic AMP concentration; in the presence of adenosine deaminase, glutamate had no effect on basal dopamine synthesis, but enhanced K(+)-stimulated synthesis. Both these effects of glutamate were abolished in Ca2(+)-free medium or in the presence of 2-amino-5-phosphonovalerate, an N-methyl-D-aspartate (NMDA) receptor blocker. In Mg2(+)-free medium with adenosine deaminase, glutamate enhanced both basal and K(+)-stimulated synthesis. These results suggest that dopaminergic terminals have A2 adenosine receptors, whose activation can stimulate dopamine synthesis by a cyclic AMP-dependent mechanism, and NMDA receptors, which modulate dopamine synthesis by a Ca2(+)-dependent mechanism.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by beta-ecdysone.

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.     

Relationship between RNA synthesis and the Ca2+-filled state of the nuclear envelope store

RNA synthesis and ATP-dependent (45)Ca(2+) uptake were measured simultaneously in isolated nuclear fraction of rat liver nuclei. Maximal level of RNA synthesis was obtained under ATP-dependent (45)Ca(2+)-uptake conditions (1 microM free [Ca(2+)] and 1 mM ATP in the bathing solution). This experimental condition was defined as "stimulated nuclei" condition. ATP-dependent (45)Ca(2+) uptake was inhibited using different strategies including: (a) eliminating Ca(2+) (1 mM EGTA); (b) lowering the ATP concentration; (c) modifying nuclear envelope membranes Ca(2+) permeability (Ca(2+) ionophores); or (d) inhibiting the nuclear Ca(2+) pump (thapsigargin and 3',3',5',5'-tetraiodophenolsulfonephthalein). Under all the above conditions, RNA synthesis was lower than in "stimulated nuclei" condition. In the presence of ionomycin, RNA synthesis was significantly higher at 500 nM free [Ca(2+)], as compared with RNA synthesis in a Ca(2+)-free medium or at 1muM free [Ca(2+)]. However, even in such condition (500 nM free [Ca(2+)]), RNA synthesis was lower than RNA synthesis obtained in "stimulated nuclei" condition. We suggest two components for the effect of Ca(2+) on RNA synthesis: (A) a direct effect of nucleoplasmic [Ca(2+)]; and (B) an effect dependent on the accumulation of Ca(2+) in the nuclear envelope store mediated by the SERCA nuclear Ca(2+) pump.     

Translational control of immunoglobulin synthesis. I. Repression of heavy chain synthesis

When myeloma cells are incubated at 25 °C the secretion of myeloma protein ceases within 20 minutes. The synthesis of heavy and light chains and the assembly into the completed 7 S immunoglobulin continue at over 40% of the synthetic rate at 37 °C, resulting in an increasing intracellular concentration of myeloma protein with time. When myeloma cells containing an increased myeloma protein pool were re-incubated at 37 °C, there was an initially decreased synthesis of H-chain² relative to L-chain or total protein. Whereas L-chain synthesis returned to the pre-25 °C synthetic rate within 15 minutes, the synthesis of H-chain required over 60 minutes to return to the pre-incubation rate.Myeloma cells maintained in exponential growth contain a larger intracellular pool of H₂L₂ than cells in late stationary phase. When both populations of cells were incubated at 25 °C and the synthesis of H and L-chain protein measured, a reduced synthesis of H-chain was again observed. Exponentially growing cells showed an 80% reduction of H-chain synthesis after 100 minutes at 25 °C. Stationary cells, with the reduced intracellular level of H₂L₂, required 210 minutes to effect an equivalent reduction of H-chain synthesis.The opposite effect on myeloma protein synthesis was observed following depletion of the H₂L₂ pool. The intracellular H₂L₂ pool was reduced by allowing secretion in the absence of protein synthesis. When protein synthesis was allowed to continue following the depletion, a stimulation of myeloma protein synthesis relative to total protein synthesis was observed.These experiments suggest a close relation between the intracellular level of H₂L₂ and the production of H-chain. From the rapidity of the repression and de-repression of H-chain synthesis, a regulation at the translational level is suggested.     

Protein Synthesis in Relation to Sporulation and Meiosis in Yeast

The dependence upon protein synthesis of physiological and biochemical events occurring during yeast sporulation was investigated. Protein synthesis was inhibited by cycloheximide. There was an early, irreversible sensitivity to inhibition with respect to cell viability and ascus formation; inhibition was reversible only if the cells were inhibited after, but not prior to, 2 to 3 h in sporulation medium. Interruption of protein synthesis of any time during sporulation inhibited all measurable metabolic and sporulation-specific processes except protein breakdown and, to some extent, ribonucleic acid synthesis. The time interval between the occurrence of an event and the protein synthesis necessary for that event was determined to be 2 to 3 h for ascus formation, 相似文献   

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear whether GLP-2 can directly stimulate protein synthesis. The objective was to test critically whether GLP-2 receptor (GLP-2R) activation directly stimulates protein synthesis through a PI 3-kinase-dependent Akt-mTOR signaling pathway. HEK 293 cells (transfected with human GLP-2R cDNA) were treated with human GLP-2 with/without pretreatment of PI 3-kinase inhibitor (LY-294002) or mTOR inhibitor (rapamycin). Results show that 1) GLP-2 specifically bound to GLP-2R overexpressed in the HEK cells with K(a) = 0.22 nM and B(max) = 321 fmol/μg protein; 2) GLP-2-stimulated protein synthesis was dependent on the amount of GLP-2R cDNA and the dosage of GLP-2 and reached the plateau among 0.2-2 nM GLP-2; 3) GLP-2-stimulated protein synthesis was abolished by the PI 3-kinase inhibitor and mTOR inhibitor; and 4) GLP-2-mediated stimulation of phosphorylation on Akt and mTOR was dependent on the amount of GLP-2R cDNA transfected and the dosage of GLP-2. In addition, GLP-2-mediated action and signaling in regulation of protein synthesis were confirmed in mouse hippocampal neurons (expressing native GLP-2R). GLP-2 directly stimulated protein synthesis of primary cultured neurons in dosage-dependent, PI 3-kinase-dependent, and rapamycin-sensitive manners, which linked with activation of Akt-mTOR signaling pathway as well. We conclude that GLP-2R activation directly stimulates protein synthesis by activating the PI 3-kinase-dependent Akt-mTOR signaling pathway. GLP-2-stimulated protein synthesis may be physiologically relevant to maintaining neuronal long-term potentiation and providing secondary mediators (namely neuropeptides or growth factors).     

Arachidonic acid regulation of prostanoid synthesis in macrophages

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.     

IL-4-based helper activity of CD4 T cells is radiation sensitive

The capacity of CD4⁺ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by γ-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2. Thus, although some functions of CD4⁺ T cells are resistant to radiation, other functions, particularly those that depend on the production of IL-4 and IL-5, are greatly diminished by ionizing radiation.     

Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP-independent pathway in osteoblastic clone MC3T3-E1 cells

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.