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Increased polyamine production is observed in a variety of chronic neuroinflammatory disorders, but in vitro and in vivo studies yield conflicting data on the immunomodulatory consequences of their production. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in endogenous polyamine production. To identify the role of polyamine production in CNS-intrinsic inflammatory responses, we defined CNS sites of ODC expression and the consequences of inhibiting ODC in response to intracerebral injection of LPS±IFNγ. In situ hybridization analysis revealed that both neurons and non-neuronal cells rapidly respond to LPS±IFNγ by increasing ODC expression. Inhibiting ODC by co-injecting DFMO decreased LPS-induced CCL2 expression and macrophage influx into the CNS, without altering LPS-induced microglial or macrophage activation. Conversely, intracerebral injection of polyamines was sufficient to trigger macrophage influx into the CNS of wild-type but not CCL2KO mice, demonstrating the dependence of macrophage influx on CNS expression of CCL2. Consistent with these data, addition of putrescine and spermine to mixed glial cultures dramatically increased CCL2 expression and to a much lesser extent, TNF expression. Addition of all three polyamines to mixed glial cultures also decreased the numbers and percentages of oligodendrocytes present. However, in vivo, inhibiting the basal levels of polyamine production was sufficient to induce expression of apolipoprotein D, a marker of oxidative stress, within white matter tracts. Considered together, our data indicate that: (1) CNS-resident cells including neurons play active roles in recruiting pro-inflammatory TREM1-positive macrophages into the CNS via polyamine-dependent induction of CCL2 expression and (2) modulating polyamine production in vivo may be a difficult strategy to limit inflammation and promote repair due to the dual homeostatic and pro-inflammatory roles played by polyamines.

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N1-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N1-acetylspermidine, putrescine, cadaverine, N1-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N1-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors at leo.hawel@ucr.edu.]

Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF–2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4α (HNF4α) activate transcription. In this study, we show that HNF4α interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4α in vivo, it did not interact well with the HNF4α ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4α and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4α and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4α-mediated transcription and that a specific inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), decreased the ability of HNF4α to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.

Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (+/-0.3 degrees C) during the initial 30 min of loose-tube restraint in conditioned animals. When conditioned/tube-trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2-3 min, ACTH increased to 83.2 +/- 31.0 pg/dl, compared to 28.1 +/- 7.7 pg/dl for cage controls, reaching a maximum at 15-30 min (254.6 +/- 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 +/- 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3-10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 +/- 54.8 compared to 31.2 +/- 10.2 pg/dl; corticosterone, 12.2 +/- 6.2 microg/dl compared to 37.1 +/- 6.4 microg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10-20 fold higher (541.8 compared to 27.2-59.1 pg/dl for ACTH) and 2-5 fold higher (45.7 compared to 8.4- 20.0 microg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5640-5648) that will identify both up- and downregulated genes differentially expressed between two cDNA populations. This manuscript provides a thorough description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers(N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. BioL 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5640-5648) is improved and requires less starting material than many existing methods. Several key modifications are included (1). Size-exclusion gel-filtration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the number of ethanol precipitations required and in improving the yield of desirable amplification products (2). Elimination of the mung bean nuclease treatment in favor of a simple dilution of PCR serves as a means of markedly reducing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninteracting primers ensures that no anomalous amplification occurs due to carryover of primers or incomplete digestion from the ends of the cDNAs (4). A set of cDNA standards was developed and various concentrations were used to better characterize the ability of representational difference analysis to identify rare messages in a complex cDNA population (5). Integral to this manuscript, a detailed laboratory protocol is available from the authors (craig.byus@ucr.edu) and provides a step-by-step description of the modified procedure.

Putrescine export was found to occur by a non-diffusional highly regulated process using Xenopus oocytes as a model system of polyamine transport. Untreated oocytes were observed to possess high endogenous intracellular putrescine and spermidine levels with no detectable polyamine interconversion or biosynthesis over the assay intervals. The putrescine uptake process demonstrated a rapid saturation within a 5 min interval. Spermidine demonstrated a relatively larger uptake capacity with only a minimal ability to export. A kinetic analysis of the concentration-dependence of the putrescine and spermidine uptake processes indicated that the putrescine uptake process may possess two concurrent uptake components while spermidine uptake may possess a two-component process with an allosteric regulation. Elevated intracellular putrescine levels were observed to decrease against a 10-fold higher extracellular concentration gradient in a rapid and specific manner. No noticeable changes in the intracellular levels of other polyamines were observed over the same time interval. The uptake and export rates of putrescine transport also showed a concurrent, rapid and cyclical regulation. These findings support a non-diffusional putrescine export process which is highly regulated.

Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acetyl-spermidine, compared with those of exported putrescine, were observed to accumulate in the culture medium. A careful analysis of the changes in the intracellular concentration of putrescine relative to the steady-state net rate of putrescine export, as the doubling time of the cultures increased from 16 h to 22 h, indicated that an inverse relationship existed between these two parameters. As the intracellular putrescine concentrations increased, the net rate of putrescine export decreased markedly. Determination of the rate of putrescine uptake indicated that putrescine uptake also decreased significantly as the cultures neared confluency, and at no time during the growth of the culture did the rate of putrescine uptake approximate to the high rate of putrescine efflux. The decrease in the putrescine export rate seen as the cells grew toward confluency was determined to be primarily due to the inhibitory effect of the effluxed putrescine in the medium (Ki = 2 microM), and not to contact inhibition. The data suggested that the efflux of putrescine and cadaverine is not mediated to a significant degree by a process involving simple diffusion.

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p, Author Summary Visceral leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is a progressive, potentially fatal infection found in many resource-poor regions of the world. We initiated these studies of an experimental model of VL to better understand the molecular and cellular determinants underlying this disease. We found that host macrophages or fibroblasts, when infected with Leishmania donovani or exposed to products secreted by the parasite, are permissive to infection because they fail to metabolize arginine to generate nitric oxide, the effector molecule needed to kill the intracellular parasites. Instead, the infected host cells are activated in a way that leads to the expression of arginase, an enzyme that metabolizes arginine to produce polyamines, which support parasite growth. This detrimental activation pathway was dependent on the parasite-induced activation of the transcription factor STAT6, but contrary to the previously accepted paradigm, did not require (but was amplified by) the presence of polarized Th2 cells or type 2 cytokines. Knockdown of host arginase or STAT6 enhanced control of the infection, indicating that this activation pathway has a critical role in the pathogenesis of the disease. Interventions designed to inhibit the STAT6-arginase-polyamine pathway could help in the treatment or prevention of VL.

We characterized the mechanism(s) involved in the efflux of putrescine/cadaverine from cultured mammalian cells using various pharmacological agents. Verapamil and quinine inhibited putrescine and cadaverine export in monocytic-leukemic RAW 264 and H35 hepatoma cells in a concentration-dependent manner with an IC50 in the micromolar range. Verapamil, which inhibits L-type calcium channels, inhibited putrescine export, regardless of whether calcium was present in the extracellular medium or not. Furthermore, the export of putrescine in the absence of verapamil did not appear to depend upon extracellular calcium. Neither intracellular calcium, external sodium, changes in intracellular pH nor phosphorylation affected the levels of putrescine export independently from changes in intracellular putrescine levels. The data suggest that verapamil and quinine inhibit putrescine/cadaverine efflux from the cell by binding directly to an integral membrane protein.

Macrophage-like RAW 264 and H35 hepatoma cells grown under serum-free conditions exported putrescine and an unidentified diamine into the culture medium. Unlike putrescine, the unknown compound could be detected only extracellularly. Analyses of dansylated polyamine standards and mass spectroscopy confirmed that the unknown compound was cadaverine (1,5-diaminopentane). The cells were free of mycoplasma as evidenced by a negative result using a probe specific for prokaryotic rRNA. After prophylactic treatments with two different mycoplasmacidal agents, the cells continued to export cadaverine. Attempts to "infect" a noncadaverine-exporting cell line with culture medium and cell-free lysates proved unsuccessful, establishing that cadaverine was in fact a bona fide product of these mammalian cells. Cadaverine export by RAW 264 and H35 cells was stimulated by lipopolysaccharide and insulin, respectively. However, administration of exogenous ornithine caused cadaverine export to decrease significantly with concomitant increases in putrescine export. alpha-Difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, inhibited both cadaverine and putrescine export. When cells were labeled with [3H]lysine, the great majority of the radioactivity recovered in exported polyamines was found in cadaverine. The cumulative data suggested that cadaverine formation may be caused by the action of intracellular ornithine decarboxylase upon lysine to produce cadaverine, which is then effluxed from the cell with a high degree of efficiency.

The cellular responses to hypoxia are poorly understood. To test the hypothesis that ornithine decarboxylase (ODC; L-ornithine carboxy-lyase; EC 4.1.1.17) activity and polyamine concentrations change in response to acute hypoxia, we performed the following studies. Pregnant Sprague-Dawley rats inspired various O2 concentrations (9-21%) for various time periods (0.5-48 h) from days 15 to 21 of gestation. In fetal brains we measured the activity of ODC, ODC mRNA, and polyamines. In response to 4-h acute mild hypoxia, ODC activity in fetal rat brain (cerebrum, cerebellum, and hippocampus) increased to 330-450% from control values (P < 0.001), after which it declined to control levels in 6-8 h. The 4-h ODC response varied inversely with inspired O2 concentration and was not mimicked by beta 2 agonist or blocked by beta 2-antagonist administration. The ODC response was associated with an increase in fetal brain putrescine concentration to 190% above control at 4-6 h (P < 0.01) and an increase in the polyamines spermidine and spermine to about 115% above control at 6-8 h. We also observed that ODC mRNA increased significantly after 2-4 h of hypoxia. ODC activity and polyamine concentrations appear to be useful enzymatic markers for fetal brain hypoxia. The magnitude and time course of the acute hypoxic ODC increase were similar to responses to extracellular signals that result in differentiation or cell growth. Thus, the well-defined and regulated ODC activity response may represent a protective mechanism in brain to hypoxia.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.

Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decaboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-β-acetate (TPA) in the presence of serum (Wu and Byus:(Biochem. Biophys. Acta 804:89–99, 1984)) Wu et al.: (Cancer Res. 41:3384–3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2–3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4–5-fold increase in intracellular steady-state ornithine levels and by a 6–8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984)) the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3–5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10−10–10−8 M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone. However, ornithine addition to the culture medium was not accompanied by any further increase in the insulin-mediated elevation in DNA synthesis. The data is discussed in relation to the selective ability of mitogens to alter the flux through extracellular and intracellular pools of ornithine as required to supply sufficient polyamines to support maximal rates of DNA synthesis. The results furthers support the suggestion that the high levels of intracellular putrescine observed following TPA + ornithine might enhance any of a number of the specific or unique transductive events employed by TPA, in comparison with insulin, which lead to the synthesis of DNA.

For a number of years, researchers have noted that cancer cells have higher levels of ornithine decarboxylase (ODC) activity than do the corresponding noncancerous tissues (1-5). Because increases in ODC activity (and subsequently, the polyamines) occur regularly in the cell cycle and are necessary before successful cell division can occur, the exact role of these increased polyamine levels in tumorigenesis was not completely understood. It was unclear whether the increases in ODC and polyamine levels directly participated in the generation on the cancer phenotype, or whether these increases were only a result of the increased cell cycling that is a hallmark of many cancerous cells and tumors (1-4).

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mM putrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.

There are two major questions undergoing considerable discussion regarding the potential biological effects elicited by exposure to electromagnetic fields. The first question is ‘Can biological systems at the subcellular or cellular level sense and respond to any of a number of environmentally relevant electromagnetic fields?’ By defining a measurable and reproducible molecular effect of exposure of a biological system to low-energy electromagnetic fields, it is hoped that the existence of a physical mechanism involved in mediating this response could be firmly established and ultimately understood. The second question of considerable interest is ‘Does exposure of the human population to these low-energy “environmentally relevant” fields pose any kind of health risk?’ The answer to this question has become particularly important due to the greater emphasis which is placed now upon the prevention rather than the treatment of disease. If, however, biological systems are incapable of sensing or responding to these fields, then there could be no health risk associated with this exposure. Furthermore, the establishment of a biological effect elicited by electromagnetic field exposure does not necessarily imply that there is any adverse effect upon the health of animals or humans.

Using human erythrocytes as a model system for the study of mammalian polyamine transport, detailed kinetic parameters regarding the uptake and export of putrescine and spermidine were determined. The putrescine uptake data indicated a multi-component uptake system comprised of a low-capacity saturable component and a non-saturable component. The saturable putrescine uptake component demonstrated a calculated Km of 21.0 microM and a V(max) of only 6.52 x 10(-13) M/s. The non-saturable linear putrescine uptake rate was defined by a significant pH dependence, a lack of uptake inhibition by related polyamines, and a permeability pi of 3.19 x 10(-8) s-1. These findings suggested that non-saturable putrescine uptake involved a process of simple diffusion. Spermidine uptake exhibited Michaelis-Menten kinetics with a Km and Vmax of 12.5 microM and 1.36 x 10(-12) M/s, respectively. Spermidine uptake did not demonstrate pH dependence and was not significantly inhibited by any of the tested polyamines. The Arrhenius plot of spermidine uptake was determined to be biphasic with calculated activation energies of spermidine uptake of 135.2 kJ/mol for 19-21 degrees C and 59.3 kJ/mol for 21-35 degrees C. These data suggest the possibility of multiple spermidine uptake processes which are not mediated by simple diffusion across the cell membrane. The putrescine export process demonstrated both saturable and non-saturable components. The calculated Km, V(max) and pi for putrescine export were 33.8 microM, 1.19 x 10(-11) M/s and 2.81 x 10(-7) s-1, respectively. The spermidine export process was non-saturable up to intracellular spermidine concentrations of 4 microM. At similar intracellular and extracellular concentrations of putrescine and spermidine, however, export processes displayed rates which were an order of magnitude greater than their respective uptake rates. This finding supports the possible presence of mediated putrescine and spermidine export processes different than simple diffusion.

Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10−7 M insulin yielded intracellular putrescine levels that remained elevated for 36 h along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0–12 h) and 1.0 (12–36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC50 approx. 1 μM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.

We have tested an 836.55 MHz field with North American Digital Cellular (NADC) modulation in a 2-year animal bioassay that included fetal exposure. In offspring of pregnant Fischer 344 rats, we tested both spontaneous tumorigenicity and the incidence of induced central nervous system (CNS) tumors after a single dose of the carcinogen ethylnitrosourea (ENU) in utero, followed by intermittent digital-phone field exposure for 24 months. Far-field exposures began on gestational day 19 and continued until weaning at age 21 days. Near-field exposures began at 35 days and continued for the next 22 months, 4 consecutive days weekly, 2 h/day. SAR levels simulated localized peak brain exposures of a cell phone user. Of the 236 original rats, 182 (77%) survived to the termination of the whole experiment and were sacrificed at age 709-712 days. The 54 rats (23%) that died during the study ("preterm rats") formed a separate group for some statistical analyses. There was no evidence of tumorigenic effects in the CNS from exposure to the TDMA field. However, some evidence of tumor-inhibiting effects of TDMA exposure was apparent. Overall, the TDMA field-exposed animals exhibited trends toward a reduced incidence of spontaneous CNS tumors (P < 0. 16, two-tailed) and ENU-induced CNS tumors (P < 0.16, two-tailed). In preterm rats, where primary neural tumors were determined to be the cause of death, fields decreased the incidence of ENU-induced tumors (P < 0.03, two-tailed). We discuss a possible approach to evaluating with greater certainty the possible inhibitory effects of TDMA-field exposure on tumorigenesis in the CNS.

Ornithine decarboxylase was purified from androgen-treated mouse kidney to homogeneity and high specific activity. The purified enzyme was utilized for production and screening of rat monoclonal and polyclonal antibodies. A rat monoclonal antibody was isolated which was capable of immunoprecipitation of native mouse kidney ornithine decarboxylase activity or the [3H]difluoromethylornithine-inactivated enzyme. Phosphorylation of mouse ornithine decarboxylase by casein kinase-II prior to immunoprecipitation led to complete loss of the epitope recognized by the monoclonal antibody but did not alter recognition by polyclonal antibody. Mammalian ornithine decarboxylase activity obtained from several species, in crude or partially purified extracts, was subjected to quantitative immunoprecipitation with monoclonal and polyclonal antibody. Polyclonal antibody immunoprecipitated all of the ornithine decarboxylase activity from every extract tested, while monoclonal antibody was capable of only limited immunoprecipitation (60-80%). Due to the inability of the monoclonal antibody to recognize ornithine decarboxylase phosphorylated in vitro by casein kinase-II and the partial immunoprecipitation of ornithine decarboxylase activity from cell extracts, a portion of the ornithine decarboxylase molecule population must exist in a phosphorylated state. This immunological evidence further confirms existing data that the enzyme exists in at least two distinct forms.

The effects of insulin and glucagon on CAMP accumulation, protein kinase activation, and glycogenolysis were investigated in isolated rat hepatocytes. Glucagon (0.01 nM to 10 μM) increased the activation state of protein kinase and the rate of glucose accumulation. Addition of 1.0 nM insulin to cells preincubated with 0.1 nM glucagon attenuated the rate of glucose accumulation, but did not alter the protein kinase activity ratio. Addition of 0.1 nM glucagon to cells pre-incubated with 1.0 nM insulin caused a rapid activation of protein kinase; however, glycogenolysis was not immediately affected. These effects were enhanced with pharmacological concentrations of glucagon and insulin. These data indicate that the degree of protein kinase activation does not always correlate temporally or quantitatively with rates of glycogenolysis in liver cells exposed to insulin and glucagon.

Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase (ODC) were studied along with RNA and DNA synthesis in human peripheral blood lymphocytes following in vitro culture with either mitogens, nonmitogenic agglutinins, or dibutyryl cyclic AMP (DBcAMP). The mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) all resulted in activation of soluble cyclic AMP-dependent protein kinase, the induction of increased levels of ODC activity, and increases in the rate of incorporation of (3H)-thymidine (DNA synthesis) and (3H)-uridine (RNA synthesis) into acid-insoluble material. High concentrations of PHA or Con A, which resulted in maximal cyclic AMP-dependent protein kinase activation, were only slightly mitogenic. The time at which maximum activation of protein kinase occurred differed for each mitogen but, in each case, always preceded ODC induction and increased RNA and DNA synthesis. In Con A-stimulated cultures, cyclic AMP-dependent protein kinase activation was maximal at 4 hr of culture and remained elevated at 24 and 48 hr. In these cultures increased ODC activity was first observed at 6 to 8 hr of culture and preceded increased RNA synthesis which was detectable at 12 hr of culture. DNA synthesis in all mitogen-stimulated cultures was first observed at 48 hr with maximum (3H)-thymidine incorporation being at 72 hr of culture. Cyclic AMP-dependent protein kinase activation, induction of ODC, and increased RNA and DNA synthesis were all shown to be dependent upon lymphocyte-Con A binding and were blocked by early addition of α-methyl mannoside. After 12 hr of culture, α-methyl mannoside addition had little effect on Con A induction of ODC or on RNA and DNA synthesis measured at 24 to 72 hr. In contrast, cyclic AMP-dependent protein kinase activation, measured at 24 hr, could be completely inhibited when α-methyl mannoside was added as late as 12 or 20 hr to Con A-stimulated cultures. The nonmitogenic agglutinins, wheat germ agglutinin (WGA) and Agaricus bisporus lectin (ABL), failed to increase any of the parameters tested. DBcAMP also did not induce ODC or cause any increased RNA or DNA synthesis. However, DBcAMP (10-3 M) did cause activation of cyclic AMP-dependent protein kinase as early as 15 min of culture. DBcAMP activation of protein kinase was concentration-dependent, maximal at 12 hr, and remained elevated through 48 hr of culture. The addition of DBcAMP (10-3 M) to Con A-stimulated cultures caused a 90 to 95% inhibition of ODC activity and RNA and DNA synthesis. This inhibition was concentration-dependent and paralleled the degree of activation of cyclic AMP-dependent protein kinase elicited by each concentration. These results suggest that the selective activation of cyclic AMP-dependent protein kinase and induction of increased ODC activity are early integral events linked to and regulating lymphocyte mitogenesis.

Summary Injection of 100μ g of 12-0-tetradecanoylphorbol-13-acetate (TPA) intraperitoneally led to a marked increase in ornithine decarboxylase (ODC) activity in the brain, liver, and lung of male rats. The enzyme activity was increased 45-fold, 9-fold, and 8-fold above controls in the liver, brain, and lung respectively. Maximal induction of the enzyme occurred at 5 hr and returned to control levels by 12 hr. Tumor promotor-induced ornithine decarboxylase activity was also observed in the liver and lung of hypophysectomised animals but not in the brain. The implications of these data on future studies involving the promotion of TPA of tumors initiated in tissues other than the epidermis are discussed.

To define the role of cAMP in the actions of ACTH, the dissociation of cAMP-dependent protein kinase and the subsequent intracellular location of its free catalytic units were monitored after exposure of Y-1 cells to ACTH, FSH, or cyclic nucleotide analog. To accomplish this, a fluorescinated cytochemical probe was used that complexes specifically with free catalytic units from cAMP-dependent protein kinase. Also, the effects of hormone or nucleotide on secretion of fluorogenic steroids and DNA synthesis were examined. Y-1 cells dissociated protein kinase in a dose-dependent fashion when exposed to ACTH or cAMP analog, but did not respond to FSH, which was one of the control agents used. After 30 min of treatment with 1.5 X 10(-10) M ACTH, free catalytic units were observed only in the cytoplasm of Y-1 cells, whereas a similar time of exposure to 3 X 10(-10) M ACTH led to the appearance of catalytic units in nucleolus as well as in cytoplasm. ACTH (6 X 10(-10) M) caused a rise in cytoplasmic and nucleolar protein kinase dissociation proportionally greater than that seen in cultures exposed to 3 X 10(-10) M ACTH. Upon treatment with 6 X 10(-10) M ACTH, the amount of free catalytic units in cytoplasm and nucleolus was detectably greater than that in controls within 1 min of stimulation and continued to rise with increasing time of exposure to hormone. The nuclear, mostly nucleolar, content of free catalytic unit appeared to peak after 15 min of stimulation, while cytoplasmic enzyme levels continued to rise up to 60 min. Exposure of Y-1 cells to nucleotide analog caused cAMP-dependent protein kinase dissociation with temporal kinetics and a subcellular distribution similar to that seen after ACTH stimulation. We conclude that actions of ACTH are mediated by cAMP-dependent protein kinases. Further, there appear to be two intracellular pools of protein kinase, one nucleolar, the other cytoplasmic, and these may be independently regulated, with the nucleolar enzyme requiring higher concentrations of ACTH for dissociation than those needed for cytoplasm protein kinase. These observations may be relevant to the fact that more ACTH is required to inhibit DNA synthesis than is necessary to enhance steroid production.

Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, may be controlled by a cAMP-dependent mechanism. This hypothesis was investigated in the adrenal medulla of the rat. Exposure of rats to cold (4°, 2 hr) leads to increased cholinergic nerve transmission and to a 10- to 20-fold increase in cAMP levels in the medulla within 30 min. The cAMP level returned to normal within 2 hr of the initiation of cold exposure. Ornithine decarboxylase activity was elevated within 1 hr of cold exposure and by 4 hr was increased 10- to 20-fold. We also studied the effects of various drugs which were agonists and antagonists of the cAMP response to cold exposure in the medulla. Aminophylline (200 μmoles/kg), an inhibitor of phosphodiesterase activity, caused a large, rapid increase in the cAMP level followed by an increase in ornithine decarboxylase activity similar to that after cold exposure. Injection of a cholinomimetic drug, carbamylcholine (4.1 μmoles/kg), caused a 10- to 15-fold increase in cAMP within 20 min and a 10-fold elevation in ornithine decarboxylase activity within 2.5 hr. Pretreatment of the rat with the nicotinic receptor antagonist, mecamylamine (15 μmoles/kg), greatly reduced the carbamylcholine-induced rise in both cAMP levels and ornithine decarboxylase activity. Mecamylamine administered alone did not alter either cAMP levels or ornithine decarboxylase activity. Administration of reserpine (16 μmoles/kg) also resulted in an early rise in cAMP concentration in the adrenal medulla and a concomitant increase of ornithine decarboxylase activity. Cyclic AMP has been postulated to exert its effect on cellular metabolism via the activation of a cAMP-dependent protein kinase. Varying doses of reserpine from 1.6 to 16 μmoles/kg yielded a 1:1 relationship between the degree of activation of cAMP-dependent protein kinase(s) and the induction of ornithine decarboxylase. We feel that evidence from this and other laboratories supports the hypothesis that ornithine decarboxylase may be controlled by cAMP-dependent protein kinase(s).

The total amounts of type I and type II cytoplasmic cyclic AMP-dependent protein kinase activities were measured in various tissues of intact rats and rats subjected to castration, hypophysectomy, or adrenalectomy. After castration, the total amount of type I activity decreased rapidly in classifically steroid-responsive tissues such as the ventral prostate and levator ani muscle and less rapidly in the liver. After hypophysectomy and adrenalectomy, type I activity in the liver decreased to the same extent as after castration. Type I activity could be maintained in the ventral prostate and levator ani muscle at control levels by the daily injection of dihydrotestosterone. Furthermore, after post-castration regression of the prostate for 3 days, three daily subcutaneous injections of dihydrotestosterone resulted in a complete restoration of type I activity toe the intact level. The amount of type II activity was not altered by any of the experimental ablations. This study provides evidence linking steroid action to the ability of steroid-responsive tissues to maintain a substantial activity of type I cyclic AMP-dependent protein kinase.

People living in the industrial society of today are unavoidably exposed to low-energy electromagnetic (EM) radiation. The potential risk to human health of such exposure has received much study. In this regard, numerous epidemiological studies have linked exposure to low-energy EM fields to increased cancer risk. We investigated the ability of low-energy 60-Hz EM fields to alter the activity of ornithine decarboxylase (ODC) in a number of established cell lines. The activity of ODC, the controlling enzyme in polyamine biosynthesis, has been shown to be elevated in growing cells or tissues and during the process of tumor promotion. A 1-h exposure to a 60-Hz EM field of an intensity of 10 mV/cm produced a 5-fold increase in ODC activity in human lymphoma CEM cells and a 2- to 3-fold increase in mouse myeloma cells (P3) relative to the unexposed cultures. Depending upon the cell type, ODC activity increased during the 1-h exposure period and remained elevated for several hours after the field exposure ended. In another series of experiments, fields of an intensity as low as 0.1 mV/cm for a 1-h period produced a 30% increase in the activity of ODC in Reuber H35 hepatoma cells grown in monolayer culture. In the H35 cells, continuous exposure to the 60-Hz EM field (10 mV/cm) for periods of 2 and 3 h resulted in either no increase in ODC activity (2 h) or a decrease in enzyme activity (3 h) compared to the unexposed control cultures. The data is discussed in relation to possible molecular mechanisms of field-cell interaction, the importance of the exposure intervals altering cellular ODC activity and the potential ability of 60-Hz EM fields to serve as a tumor promoting stimulus.

It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.

The administration of aminophylline results in rapid increases in cyclic AMP in the adrenal medulla, adrenal cortex, liver, and kidney of the rat. The injection of theophylline results in a similar increase in cyclic AMP in the liver of the rat. In all instances, these increases are followed by 4- to 2-fold elevations of ornithine decarboxylase activity. The generality of this phenomena suggests that ornithine decarboxylase activity is regulated by an increase in cyclic AMP.

The induction or ornithine decarboxylase activity ( l -ornithine carboxy-lyase, EC 4.1.1.17) by either 8-bromoadenosine 3′,5′ cyclic monophosphate (8Br-cAMP) or the tumor promoting phorbol ester, 12- O -tetradecanoyl-13-acetate (TPA) was studied in a series of Chinese hamster ovary (CHO) cell mutants deficient in the enzyme cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). 8Br-cAMP induced ornithine decarboxylase activity in the parental or wild-type cell line (10001) in a dose-dependent manner, with 1 mM 8Br-cAMP causing a 10–12-fold increase in ornithine decarboxylase activity, which was maximal at 2–3 h following addition of the analog. Similar treatment with 1 mM 8Br-cAMP caused only a 0–3-fold increase in ornithine decarboxylase activity in four cAMP-dependent protein kinase mutants studied (10215, 10248, 10260, 10265), indicating that functional cAMP-dependent protein kinase is required for the 8Br-cAMP induced increase in ornithine decarboxylase activity. The tumor promoting phorbol ester, TPA (1.6 μM), led to a 10–15-fold increase in ornithine decarboxylase activity in the parental cell line (10001) with maximal levels reached at 3–4 h following addition of the agent. In three out of four cAMP-dependent protein kinase mutants, 1.6 μM TPA caused an 8–10-fold increase in ornithine decarboxylase activity, demonstrating that TPA caused an elevation in ornithine decarboxylase activity by a mechanism not totally dependent upon a functional cAMP-dependent protein kinase system. Both 8Br-cAMP and TPA were capable of stimulating the accumulation of putrescine (the product of ornithine decarboxylase) in the wild-type cell line (10001) at 6 h following treatment. However, only TPA was capable of stimulating an accumulation of putrescine in the cAMP-dependent protein kinase mutants. Neither 8Br-cAMP nor TPA increased the level of spermidine or spermine at 6, 10 or 24 h following treatment in either the parental cell line or cAMP-dependent protein kinase mutants.

Both exposure to cold and administration of aminophylline result in rapid increases in cyclic adenosine monophosphate (cyclic AMP) in the adrenal medulla and adrenal cortex. These increases are followed by dramatic increases in ornithine decarboxylase activity is due to new enzyme systhesis. The data suggest that the decarboxylase activity is regulated by an increase in cyclic AMP.

The parenteral administration of a single dose of 3-methylcholanthrene to rats caused an increase in the liver of the concentration of 3', 5'-cAMP and of the activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). These events were followed by an increased activity of ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17), the enzyme that controls the biosynthesis of polyamines. Finally, the activity of benzo[a]pyrene hydroxylase, as well as the amount of cytochrome P-448, was increased. Similarly, after the administration of phenobarbital, there was first an increase in the cAMP concentration and in the activity of cAMP-dependent protein kinase, then the induction of ornithine decarboxylase, and finally, an enhanced activity of ethylmorphine N-demethylase and an increased content of cytochrome P-450. These data suggest that the drug-induced processes in liver that increase the activities of the oxidative, and presumably other, drug-metabolizing enzymes include the following sequence of events: (1) increase in cAMP concentration and/or activation of cAMP-dependent protein kinase; (2) induction of ornithine decarboxylase; and, (3) induction of drug-metabolizing enzymes.

We investigated the temporal relationship between the level of cyclic AMP, the activation of cyclic AMP-dependent protein kinase(s), and the induction of ornithine decarboxylase in two important rapid growth systems: the regenerating rat liver and the remaining adrenal gland following unilateral adrenalectomy. There was a biphasic increase in the aactivity of ornithine decarboxylase at 4 and 14 h following partial hepatectomy. The concentration of cyclic AMP increased 2-fold compared to sham-operated animals within 2–3 h, returned to baseline by 8 h, and was elevated again 3-fold by 12 hours. The activation of cyclic AMP-dependent protein kinase(s) occured in a similar biphasic manner. From a control activity ratio (−cAMP/+cAMP) of 0.4, values for total soluble kinase activation reached 0.75 at both 2 and 14 h. After a delay of 2 h following unilateral adrenalectomy, ornithine decarboxylase activity in the remaining adrenal gland increased 15–20-fold of control level by 8 h. Cyclic AMP concentrations in the adrenal were elevated 3.5-fold within 30 min. The protein kinase activation increased from 0.25 to nearly a totally activated state of 1.0 within 1 h, decline to 0.4 by 2 h, and returned to the level of the sham-operated controls at 8 h. In both the rat liver in response to partial hepatectomy and the adrenal gland undergoing hypertrophy, cyclic AMP-dependent protein kinase(s) was markedly activated prior to increases in ornithine decarboxylase activity.

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC50 was estimated to be about 2.63 × 10−7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.

Publisher Summary The fluoresceinated protein kinase inhibitor protein (F-PKI) procedure has been used to examine the kinetics of cAMP-dependent protein kinase dissociation and subsequent changes in the amount and subcellular distribution of free C subunits in a variety of cells. Due to the unique ability of the F-PKI probe to bind only to the free catalytic subunit this procedure offers the additional advantage of allowing the investigator to monitor the degree of activation of cAMP-dependent protein kinase(s) in discrete cells in a mixed cell or tissue population. In all cell cultures, whether clonal or primary, thus far examined there has been notable heterogeneity in the amount of F-PKI bound among cells of a given culture. Similar, though less distinct unequal binding of F-PKI also occurs in tissues. This heterogeneity occurs even when cultures are exposed to 8-Br-cAMP, suggesting that it is due to differences in the physiologic status among cells. In support of this, it is found that there is a greater uniformity of response to 8-Br-cAMP stimulation when Reuber H-35 cells are cultured in serum-free medium for 48 hr prior to their exposure to nucleotide.

The ability of the phorbol-ester tumor promoters to alter ornithine decarboxylase (ODC) activity in the liver of the rat and mouse was determined. The injection of 12-O-tetra-decanoyl phorbol-13-acetate (TPA, 100 microgram, i.p.) led to a 250-fold increase in hepatic ODC activity within 4 h of administration. This increase in ODC activity required both RNA and protein synthesis and did not occur when a variety of the non-tumor promoting phorbol-ester derivatives were administered to the rat. A distinct dose-dependent increase in hepatic ODC activity could be observed at 4 h following the injection of increasing amounts of TPA (0-100 microgram, i.p.). As little as 1.0 microgram TPA (i.p.) administered to a rat resulted in a significant stimulation in the activity of ODC in the liver compared to the control unstimulated values. Both 200 micrograms and 500 micrograms TPA produced less of an elevation in hepatic ODC activity than did the optimal dose of 100 micrograms. In the mouse, the administration of 1 microgram and 20 micrograms of TPA (i.p.) both led to a marked increase in hepatic ODC activity at 7 h and 4 h, respectively, following injection. A 4-5-fold increase in putrescine levels occurred in the rat liver in a biphasic manner between 4-8 h and 16-24 h following the injection of TPA (100 micrograms). No alterations in either spermidine or spermine were observed during this period. The administration of 100 micrograms of TPA to the rat did not alter the incorporated control animals. Under these identical conditions partial hepatectomy led to a large increase in DNA synthesis.

In 1959 Furshpan and Potter reported the presence of electrical transmission at the crayfish giant motor synapse. Soon thereafter a similar means of cell-cell communication was described for Mauthner neurons of the goldfish brain (Furshpan and Furakawa, 1962) and for mammalian smooth muscle (Dewey and Barr, 1962). In each instance contacting cells, when impaled with microelectrodes, were found to exchange ions, (perhaps K+, Na+, Cl-) through low resistance pathways connecting adjacent cytoplasms without using extracellular routes. This ionic (or electrotonic) coupling was bidirectional and occurred with an impedance 3–4 orders of magnitude less than that of the transmembrane potential. Since these observations were made in excitable tissues (nerve and muscle) their functional significance was easily reconciled (Bennett et al., 1963).

We have studied the correlation between cAMP-dependent protein kinase activation and rates of glycogenolysis in hepatocytes isolated from fed rats. With doses of 20 μM glucagon, the protein kinase was activated to a -cAMP/+cAMP ratio of 0.8 within 10 min and remained activated for up to 2 hours. A dose-response relationship between protein kinase activation and rates of glycogenolysis can be demonstrated to 0–20 μM glucagon. Glycogenolysis was stimulated greater than 2-fold after 2 hours of incubation with the higher doses of glucagon. Protein kinase activity ratios correlated well with the rates of glycogenolysis as the ratios varied from control levels of about 0.25 to the stimulated values of 0.5–0.6. However, as the ratios increased from 0.6 to 0.8, with higher doses of glucagon, there were no corresponding increases in the rates of glycogenolysis. These data may indicate (1) that activation of all of the protein kinase present in the liver cells is not necessary for maximal stimulation of glycogenolysis, or (2) that a specific protein kinase is involved in the intracellular control of glycogen breakdown in isolated rat hepatocytes.

The amount of type I and type II cyclic AMP-dependent protein kinase present in the rat heart was determined at various times during isoproterenol-induced cardiac hypertrophy. Wistar rats were injected twice daily with isoproterenol (5 mg/kg, s.c.) for 2, 5 or 10 days. Cardiac weight increased gradually over the 10-day period of drug administration, and by day 10, heart weight was 156% of control. Following the cessation of isoproterenol administration, the cardiac weight regressed toward the control value by day 15. An increase in the specific activity of type I protein kinase to 197% of control occurred by day 10. The specific activity of type II protein kinase did not change significantly during either the hypertrophy or regression stage. The increase in the specific activity of type I protein kinase during a chemically-induced trophic response of the heart may indicate that type I cyclic AMP-dependent protein kinase plays a regulatory function in this process.

THE role of cyclic AMP in the regulation of cell growth and differentiation has been extensively investigated in many cell types following various growth stimuli1,2. In recent years, peripheral blood lymphocytes treated with antigens3, plant lectins4, lipopolysaccharides5, or periodate oxidation6 have served as a model system for assessing whether cyclic AMP is involved in the regulation of the mitogenic response promoted in these cells by the above classes of agents7,8. There are two conflicting hypotheses, as outlined by Friedman1, concerning the role of cyclic AMP in lymphocyte proliferation. The first hypothesis suggests that the increase in cyclic AMP levels following mitogen stimulation8 and the subsequent increase in 32P incorporated into F1 histones9 and certain cystosol proteins10 implicates cyclic AMP as a positive mediator of mitogenesis in lymphocytes. The other hypothesis reasons that cyclic AMP inhibits proliferation, and this hypothesis is supported primarily by observations from several laboratories that raising the intracellular cyclic AMP level through the use of phosphodiesterase inhibitors11, prostaglandins12, or cyclic AMP analogues11,13–15 effectively inhibits mitogen-stimulated RNA and DNA synthesis. We present here evidence that may help resolve this apparent paradox. Incubation of Ficoll–Hypaque purified human peripheral blood lymphocytes with concanavalin A (conA) leads to the activation of only type I cyclic AMP-dependent protein kinase in these cells even though there are significant amounts of both type I and type II kinase present. But, the addition of dibutyryl cyclic AMP (DBcAMP) to the conA-stimulated lymphocytes at a concentration sufficient to block the synthesis of RNA and DNA results in the activation of both type I and type II cyclic AMP-dependent protein kinase. It therefore seems likely that while the activation of type I protein kinase represents a positive component in the progression of events promoted in a lymphocyte by a mitogenic signal (as it does in other cell types in which a trophic response is evoked), the activation of type II protein kinase (or perhaps types I and II in concert) represents a mechanism by which a negative influence can be imposed on the proliferative process by the generation of abnormally high levels of intracellular cyclic AMP.