Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-μT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis. Bioelectromagnetics 19:388–391, 1998. © 1998 Wiley-Liss, Inc.     

Resting EEG is affected by exposure to a pulsed ELF magnetic field

An increasing number of reports have demonstrated a significant effect of extremely low frequency magnetic fields (ELF MFs) on aspects of animal and human behavior. Recent studies suggest that exposure to ELF MFs affects human brain electrical activity as measured by electroencephalography (EEG), specifically within the alpha frequency (8-13 Hz). Here we report that exposure to a pulsed ELF MF with most power at frequencies between 0 and 500 Hz, known to affect aspects of analgesia and standing balance, also affects the human EEG. Twenty subjects (10 males; 10 females) received both a magnetic field (MF) and a sham session in a counterbalanced design for 15 min. Analysis of variance (ANOVA) revealed that alpha activity was significantly higher over the occipital electrodes (O1, Oz, O2) [F(1,16) = 6.858; P =.019, eta2 = 0.30] and marginally higher over the parietal electrodes (P3, Pz, P4) [F(1,16) = 4.251; P =.056, eta2 = 0.21] post MF exposure. This enhancement of alpha activity was transient, as it marginally decreased over occipital [F(1,16) = 4.417; P =.052; eta2 = 0.216] and parietal electrodes [F(1,16) = 4.244; P =.056; eta2 = 0.21] approximately 7 min after MF exposure compared to the sham exposure. Significantly higher occipital alpha activity is consistent with other experiments examining EEG responses to ELF MFs and ELF modulated radiofrequency fields associated with mobile phones. Hence, we suggest that this result may be a nonspecific physiological response to the pulsed MFs.     

Exposure to 50 Hz magnetic field modulates GABAA currents in cerebellar granule neurons through an EP receptor‐mediated PKC pathway

Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF‐MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ‐aminobutyric acid (GABA) type A receptors (GABA_ARs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF‐MF on GABA_ARs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF‐MF for 60 min. significantly increased GABA_AR currents without modifying sensitivity to GABA. However, activation of PKA by db‐cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field‐induced‐increase of GABA_AR currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF‐MF exposure, which was subsequently blocked by application of DHA or EP1 receptor‐specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2‐EP4 receptor‐specific antagonists. SC19220 also significantly inhibited the ELF‐MF‐induced elevation on GABA_AR currents. Together, these data obviously demonstrated for the first time that neuronal GABA_A currents are significantly increased by ELF‐MF exposure, and also suggest that these effects are mediated via an EP1 receptor‐mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects.     

Possible mechanisms of synaptic plasticity modulation by extremely low-frequency magnetic fields

Understanding the biological mechanisms by which extremely low-frequency (ELF, < 300 Hz) magnetic fields (MFs) interact with human brain activity is an active field of research. Such knowledge is required by international agencies providing guidelines for general public and workers exposure to ELF MFs (such as ICNIRP, the International Commission on Non-Ionizing Radiation Protection). The identification of these interaction mechanisms is extremely challenging, since the effects of ELF MF exposure need to be monitored and understood at very different spatial (from micrometers to centimeters) and temporal (from milliseconds to minutes) scales. One possibility to overcome these issues is to develop biophysical models, based on the systems of mathematical equations describing the electric or metabolic activity of the brain tissue. Biophysical models of the brain activity offer the possibility to simulate how the brain tissue interacts with ELF MFs, in order to gain new insights into experimental data, and to test novel hypotheses regarding interaction mechanisms. This paper presents novel hypotheses regarding the effects of power line (60 Hz in North America) MFs on human brain activity, with arguments from biophysical models. We suggest a hypothetic chain of events that could bridge MF exposure with detectable effects on human neurophysiology. We also suggest novel directions of research in order to reach a convergence of biophysical models of brain activity and corresponding experimental data to identify interaction mechanisms.     

Repetitive exposure to a 60-Hz time-varying magnetic field induces DNA double-strand breaks and apoptosis in human cells

We investigated the effects of extremely low frequency time-varying magnetic fields (MFs) on human normal and cancer cells. Whereas a single exposure to a 60-Hz time-varying MF of 6 mT for 30 min showed no effect, repetitive exposure decreased cell viability. This decrease was accompanied by phosphorylation of γ-H2AX, a common DNA double-strand break (DSB) marker, and checkpoint kinase 2 (Chk2), which is critical to the DNA damage checkpoint pathway. In addition, repetitive exposure to a time-varying MF of 6 mT for 30 min every 24 h for 3 days led to p38 activation and induction of apoptosis in cancer and normal cells. Therefore, these results demonstrate that repetitive exposure to MF with extremely low frequency can induce DNA DSBs and apoptosis through p38 activation. These results also suggest the need for further evaluation of the effects of repetitive exposure to environmental time-varying MFs on human health.     

Mechanism and regulation of neutrophil priming by platelet-activating factor

Although a weak direct stimulus of superoxide anion (O₂^?) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley-Liss, Inc.     

Sex and estrous cycle differences in the behavioral effects of high-strength static magnetic fields: role of ovarian steroids

Advances in magnetic resonance imaging are driving the development of higher-resolution machines equipped with high-strength static magnetic fields (MFs). The behavioral effects of high-strength MFs are largely uncharacterized, although in male rats, exposure to 7 T or above induces locomotor circling and leads to a conditioned taste avoidance (CTA) if paired with a novel taste. Here, the effects of MFs on male and female rats were compared to determine whether there are sex differences in behavioral responses and whether these can be explained by ovarian steroid status. Rats were given 10-min access to a novel saccharin solution and then restrained within a 14-T magnet for 30 min. Locomotor activity after exposure was scored for circling and rearing. CTA extinction was measured with two-bottle preference tests. In experiment 1, males were compared with females across the estrous cycle after a single MF exposure. Females circled more and acquired a more persistent CTA than males; circling was highest on the day of estrus. In experiment 2, the effects of three MF exposures were compared among intact rats, ovariectomized females, and ovariectomized females with steroid replacement. Compared with intact rats, ovariectomy increased circling; estrogen replacement blocked the increase. Males acquired a stronger initial CTA but extinguished faster than intact or ovariectomized females. Thus the locomotor circling induced by MF exposure was increased in females and modulated by ovarian steroids across the estrous cycle and by hormone replacement. Furthermore, female rats acquired a more persistent CTA than male rats, which was not dependent on estrous phase or endogenous ovarian steroids.     

EXPOSURE TO 50 Hz MAGNETIC FIELDS DOES NOT INDUCE THE PHOSPHORYLATION OF SEK1/MKK4 IN CULTURED CELLS

In our previous studies, we found that 50 Hz magnetic fields (MFs) could induce the phosphorylation of stress-activated protein kinase (SAPK) and enhance its enzymatic activity. In order to clarify the relationship between MF exposure and the SAPK pathway clearly, we studied the effects of 50 Hz MF exposure on phosphorylation (activation) of SEK1/MKK4 (the upstream kinase of SAPK). A Chinese hamster lung (CHL) cell line was exposed to 50 Hz MFs at two intensities (0.4 and 0.8 mT) for different durations, and Western blot analysis was used to measure the degree of phosphorylation (activation), and nonphosphorylation (non-activation) of SEK1/MKK4 with corresponding antibodies. The results showed that the exposures at both intensities could not induce the phosphorylation of SEK1/MKK4. However, treatment with high osmotic pressure NaCl could induce the phosphorylation of SEK1/MKK4 in cultured cells. It is suggested that 50 Hz MFs may activate the SAPK through a kinase other than SEK1/MKK4.     

Protein kinase C in erythroid and megakaryocytic differentiation: possible role in lineage determination

Erythroid differentiation of normal human hematopoietic progenitor cells was drastically inhibited by phorbol ester, 12-myristate 13-acetate (PMA), an agent known to activate the class of serine-threonine kinases, protein kinase C (PKC). This inhibition was accompanied by augmented megakaryocytic differentiation as demonstrated by expression of megakaryocyte-specific mRNAs and proteins. These effects of PMA were reversed by two specific antagonists of PKC. Analysis of single colonies transferred from cultures not containing PMA to PMA-containing cultures indicated that, in this system, PMA exerts megakaryocytic differentiating activity directly on cells which may have already initiated a progression toward the erythroid pathway of differentiation. These results suggest that modulation of PKC activity plays a role in erythroid and megakaryocytic differentiation, and may constitute an important selective signal between these pathways during normal blood cell development.     

Effects of low-frequency magnetic fields on fetal development in CBA/Ca mice

Effects of alternating magnetic fields (MFs) on the embryonic and fetal development in CBA/Ca mice were studied. Mated females were exposed continuously to a sinusoidal 50 Hz (13 μT or 0.13 mT root mean square) or a sawtooth 20 kHz (15 μT peak-to-peak) MF from day 0 to day 18 of pregnancy for 24 h/day until necropsied on day 18. Control animals were kept under the same conditions without the MF. MFs did not cause maternal toxicity. No adverse effects were seen in maternal hematology and the frequency of micronuclei in maternal bone marrow erythrocytes did not change. The MFs did not increase the number of resorptions or fetuses with major or minor malformations in any exposure group. The mean number of implantations and living fetuses per litter were similar in all groups. The corrected weight gain (weight gain without uterine content) of dams, pregnancy rates, incidences of resorptions and late fetal deaths, and fetal body weights were similar in all groups. There was, however, a statistically significant increase in the incidence of fetuses with at least three skeletal variations in all groups exposed to MFs. In conclusion, the 50 Hz or 20 kHz MFs did not increase incidences of malformations or resorptions in CBA/Ca mice, but increased skeletal variations consistently in all exposure groups. Bioelectromagnetics 19:477–485, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester.

Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF-kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.     

Interaction of palmitoylcarnitine with protein kinase C in neuroblastoma NB-2a cells

As reported previously [Acta Neurobiol. Exp. 57 (1997) 263], palmitoylcarnitine was observed to promote differentiation of neuroblastoma NB-2a cells with a concomitant inhibition of proliferation and of the phorbol ester stimulated activity of the protein kinase C (PKC). In the present study, palmitoylcarnitine was observed to inhibit phosphorylation of the PKC peptide substrate and to completely diminish binding of phorbol 12-myristate-13-acetate (PMA), although the effect was found to be uncompetitive. The exposure of NB-2a cells to palmitoylcarnitine in the presence of PMA resulted in a dramatic decrease in phosphorylation of the conventional and novel isozymes of PKC, mainly on serine. This effect was observed to be dose dependent. Inhibitors of serine/threonine phosphatases were not influencing the effect of palmitoylcarnitine what can point to an interaction between PKC and palmitoylcarnitine, affecting the process of autophosphorylation. These findings suggest that pamitoylcarnitine could be a natural modulator of PKC activity, thus regulating the process of cell differentiation.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.     

Time-varying magnetic fields of 60 Hz at 7 mT induce DNA double-strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.     

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5–1.000 mV/cm p-p) were applied for 1 h to cells (10⁷/ml) in Teflon chambers at 37 °C in the presence or absence of 2 μM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100–1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA. EFs (33–330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents. © 1996 Wiley-Liss, Inc.     

Effect of phorbol and bryostatin I on chondrogenic expression of chick limb bud, in vitro

The ability of phorbol esters to promote tumor formation and alter cell differentiation has been attributed to its action on a number of critical cellular functions, in particular, on protein phosphorylation, through the activation of protein C kinase. The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10(-7) M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression, i.e., that other pathway(s) must also be involved. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early (0-48 h) exposure to PMA (during chondrocytic commitment in vitro) had little inhibitory effect on the staining index, whereas, exposure from 49-96 h (presumably post-commitment) and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to our knowledge of the control processes involved in tumor promotion and cell differentiation.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.