Leo Hawel

Identification and characterization of a diamine exporter in colon epithelial cells.

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.

Effect of immobilization and concurrent exposure to a pulse-modulated microwave field on core body temperature, plasma ACTH and corticosteroid, and brain ornithine decarboxylase, Fos and Jun mRNA.

Abstract Stagg, R. B., Hawel, L. H., III, Pastorian, K., Cain, C., Adey, W. R. and Byus, C. V. Effect of Immobilization and Concurrent Exposure to a Pulse-Modulated Microwave Field on Core Body Temperature, Plasma ACTH and Corticosteroid, and Brain Ornithine Decarboxylase, Fos and Jun mRNA. 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°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 μg/dl compared to 37.1 ± 6.4 μg/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 μg/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.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

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<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Selective putrescine export is regulated by insulin and ornithine in Reuber H35 hepatoma cells

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 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 microM) 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.

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

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 N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-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 N(1)-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.]

Regional variation of the inwardly rectifying potassium current in the canine heart and the contributions to differences in action potential repolarization

The inward rectifier potassium current, IK1, contributes to the terminal phase of repolarization of the action potential (AP), as well as the value and stability of the resting membrane potential. Regional variation in IK1 has been noted in the canine heart, but the biophysical properties have not been directly compared. We examined the properties and functional contribution of IK1 in isolated myocytes from ventricular, atrial and Purkinje tissue. APs were recorded from canine left ventricular midmyocardium, left atrial and Purkinje tissue. The terminal rate of repolarization of the AP in ventricle, but not in Purkinje, depended on changes in external K(+) ([K(+)]o). Isolated ventricular myocytes had the greatest density of IK1 while atrial myocytes had the lowest. Furthermore, the outward component of IK1 in ventricular cells exhibited a prominent outward component and steep negative slope conductance, which was also enhanced in 10 mM [K(+)]o. In contrast, both Purkinje and atrial cells exhibited little outward IK1, even in the presence of 10 mM [K(+)]o, and both cell types showed more persistent current at positive potentials. Expression of Kir2.1 in the ventricle was 76.9-fold higher than that of atria and 5.8-fold higher than that of Purkinje, whereas the expression of Kir2.2 and Kir2.3 subunits was more evenly distributed in Purkinje and atria. Finally, AP clamp data showed distinct contributions of IK1 for each cell type. IK1 and Kir2 subunit expression varies dramatically in regions of the canine heart and these regional differences in Kir2 expression likely underlie regional distinctions in IK1 characteristics, contributing to variations in repolarization in response to in [K(+)]o changes.

LPS-INDUCED CCL2 EXPRESSION AND MACROPHAGE INFLUX INTO THE MURINE CENTRAL NERVOUS SYSTEM IS POLYAMINE-DEPENDENT

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.

Additional considerations about the bioeffects of mobile communications

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.