Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway

Abstract

Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

Figure 1. Time-dependent increase in I _Na in cerebellar GCs following exposure to ELF-EMF (1 mT or 0.4 mT).

Superimposed I _Na evoked by a 20 ms depolarizing pulse from a holding potential from −100 to −20 mV. Current traces were obtained from cerebellar GCs exposed to ELF-EMF (1 mT) for lengths of time ranging from 10 min to 90 min. (B) Statistical analysis of the activating effects of ELF-EMF (1 mT) exposure at various times on the density of I _Na. The data are reported as the mean ± S.E.M. from 8–16 cells. *, P<0.05 compared to control using a one-way ANOVA test. (C) I _Na traces obtained from cerebellar cells exposed to ELF-EMF (0.4 mT) for 6 h and 12 h. (D) Statistical analysis of the activating effects of ELF-EMF (0.4 mT) exposure for 6 h and 12 h on the density of I _Na. The data are reported as the mean ± S.E.M. from 8–10 cells. *, P<0.05 compared to control using Student’s t-test.

Figure 2. The effects of 1 mT ELF-EMF exposure on the steady-state activation and inactivation of I _Na.

(A) The effects of 60 minutes of ELF-EMF exposure on the steady-state activation of I _Na. The cells were held at −100 mV and depolarized in 5 mV steps from −70 to 20 mV with intervals of 5 s. (B) The voltage-dependent activation curve of I _Na in control cells and cells exposed to ELF-EMF. The data are from 13 (control) or 12 (ELF-EMF-exposed) cells and are expressed as means ± SEM. (C) Comparison of the plot of the normalized conductance of I _Na as a function of the command potential in control and ELF-EMF-exposed cells. (D) The effects of 60 minutes of ELF-EMF exposure on the steady-state inactivation of I _Na. The voltage protocol is shown below the current record. (E) Steady-state inactivation curves of I _Na for control and ELF-EMF-exposed cells. The data are from 6 (control) and 5 cells (ELF-EMF-exposed) and are expressed as means ± S.E.M.

Figure 3. Involvement of the PKA pathway in the increase in I _Na induced by ELF-EMF exposure.

(A) Current traces show the inhibitory effects of the selective PKA antagonist H-89 and db-cAMP on the increase in I _Na induced by a 60-minute exposure to a 1 mT ELF-EMF. (B) Statistical analysis of the inhibitory effects of H-89 and db-cAMP on the increase in I _Na density induced by a 60-minute exposure to a 1 mT ELF-EMF. The data are reported as the mean ± S.E.M. from 10–12 cells. *, P<0.05 compared to control (non-ELF-EMF group) using a Student’s t-test. #, P<0.05 compared to the corresponding control (without H-89 and db-cAMP) non-ELF-EMF group using a Student’s t-test. (C) Western blot analysis of the effects of ELF-EMF exposure on cellular pPKA levels. Upper panels show representative samples; the statistical analysis is shown in the lower panels; *, P < 0.05 compared to the corresponding control using Student’s t-test.

Figure 4. ELF-EMF exposure increased the Na_V1.2 α-subunit on the membrane in cerebellar GCs.

(A) Cells expressing Na_V 1.2 were labeled with Na_V1.2 specific antibody (red one) and transfected GC cells showing strong eGFP expression (left, see arrowhead). Cells transfected with siRNA vectors (same cell in the right, see arrowhead) showed dramatic reduction in Na_V 1.2 expression. Scale bar was 10 µM. (B) The current recordings in a control cell and a post-transfected cell of Na_V 1.2 siRNA plus eGFP. Current evoked by a 20 ms depolarizing pulse from a holding potential of −100 to −20 mV. (C) Na_V 1.2 protein on membrane surface (Na_V 1.2 M) was detected with the biotinylation assay after 30 or 60-minute exposure to a 1 mT ELF-EMF. Upper panels show representative samples, TFR (transferrin) was used as the loading control; the statistical analysis is shown in the lower panels. *, P< 0.05 compared to the corresponding control using Student’s t-test. (D) Western blot analysis of the total level of Na_V 1.2 expression (Na_V 1.2 T) after 30 or 60-minute exposure to a 1 mT ELF-EMF.

Figure 5. Effects of EP receptor antagonists on the increase in I _Na density induced by ELF-EMF exposure.

(A) Current traces show the blocking effect of the EP2 and EP4 receptor antagonists AH6809 and AH23848 on the increase in I _Na induced by a 60-minute exposure to a 1 mT ELF-EMF. (B) Statistical analysis of the inhibitory effect of AH6809 and AH23848 on the I _Na density increase induced by a 60-minute exposure to a 1 mT ELF-EMF. #, P<0.05 compared to the corresponding control (non-ELF-EMF) using Student’s t-test. *, P<0.05 compared to ELF-EMF exposure alone using a Student’s t-test. (C) Western blot analysis of the inhibitory effect of AH6809 and AH23848 on intracellular PKA phosphorylation induced by a 60-minute exposure to a 1 mT ELF-EMF. Upper panels show representative samples; the statistical analysis is shown in the lower panels; *, P< 0.05 compared to the corresponding control using Student’s t-test.

Figure 6. Effects of the AA/PGE₂ pathway on the ELF-EMF exposure-induced increase in I _Na in cerebellar GCs.

(A) PGE₂ release from cerebellar GCs exposed to 1 mT ELF-EMF for various lengths of time. The data are from three and four independent experiments, respectively. *, P< 0.05 compared to the corresponding control using Student’s t-test. (B) Intracellular AA levels in cerebellar GCs exposed to 1 mT ELF-EMF for various lengths of time. The data are from three and four independent experiments, respectively. *, P< 0.05 compared to the corresponding control using Student’s t-test. (C) Current traces show the inhibitory effects of the COX-2 inhibitors flufenamaic acid (FA) and niflumic acid (NA) on the increase in I _Na induced by 60 minutes of exposure to a 1 mT ELF-EMF. (D) Statistical analysis of the effects of FA and NA on the increase in I _Na induced by a 60-minute exposure to a 1 mT ELF-EMF. (E) Statistical analysis of the effects of FA and NA on PGE₂ release induced by a 60-minute exposure to a 1 mT ELF-EMF.

Figure 7. Effects of exposure to a 1 mT ELF-EMF on intracellular COX-1, COX-2 and cPLA₂ activities in cerebellar GCs.

(A and B) COX-1 and COX-2 activity were measured in cerebellar GCs exposed to a 1 mT ELF-EMF for various lengths of time. The data are from three and four independent experiments, respectively. (C) cPLA₂ activity was measured in cerebellar GCs exposed to a 1 mT ELF-EMF for various lengths of time. *, P<0.05 compared to the corresponding control using a Student’s t-test. (D) Immunostaining showing the effects of ELF-EMF exposure on intracellular cPLA₂ levels. The scale bar represents 20 µm.

Figure 8. A proposed model depicting the mechanisms that are likely to be involved in the modulation of I _Na by ELF-EMF exposure in cerebellar GCs.

ELF-EMF active cPLA₂ and up-regulated AA and PGE2, which can act in an autocrine or paracrine manner to activate EP receptors. Ligand binding of EP2/4 is associated with PKA activation and consequently modulates I _Na. (+), activation; (−), inhibition.