GDF-15 enhances intracellular Ca2+ by increasing Cav1.3 expression in rat cerebellar granule neurons.

GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-ß superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K(+) currents and Kv2.1 α subunit expression through TßRII (TGF-ß receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca(2+) concentration ([Ca(2+)]i) in response to membrane depolarization, as determined by Ca(2+) imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca(2+) current (ICa) without altering steady-state activation of Ca(2+) channels. Treatment with nifedipine, an inhibitor of L-type Ca(2+) channels, abrogated GDF-15-induced increases in [Ca(2+)]i and ICa The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TßRII phosphorylation. Given that Cav1.3 is not only a channel for Ca(2+) influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TßRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons.

cAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE2-Induced Increases in rNaV1.4 Current.

Arachidonic acid (AA) and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNaV1.4 Na+ current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNaV1.4 current peak without modulating the steady-state activation and inactivation properties of the rNaV1.4 channel. These results differed from the effects of extracellular application of AA on rNaV1.4 current. The effects of intracellular AA were mimicked by prostaglandin E2 but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE2-induced activation of rNaV1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE2 receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE2-induced increase of rNaV1.4 current. Two mutated channels, rNaV1.4S56A and rNaV1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE2-mediated regulation of rNaV1.4 currents. In rNaV1.4S56A, the effects of intracellular db-cAMP, AA, and PGE2 were significantly reduced. The results of the present study suggest that intracellular AA augments rNaV1.4 current by PGE2/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.

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.

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.

Arachidonic acid modulates Na+ currents by non-metabolic and metabolic pathways in rat cerebellar granule cells.

AA (arachidonic acid), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of Na(V) (voltage-gated Na(+) channels) in rat cerebellar GCs (granule cells). The extracellular application of AA inhibited the resultant I(Na) (Na(V) current), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the GCs with ETYA (eicosa-5,8,11,14-tetraynoic acid) or a membrane-impermeable analogue of AA, AA-CoA (arachidonoyl coenzyme A), we inferred that AA itself exerted the observed modulatory effects on I(Na). In contrast, intracellular AA significantly augmented the elicited I(Na) peak when the same protocol that was used for extracellular AA was followed. The observed I(Na) increase that was induced by intracellular AA was mimicked by the AA cyclo-oxygenase metabolite PGE(2) (prostaglandin E(2)), but not by ETYA. Furthermore, cyclo-oxygenase inhibitors decreased I(Na) and quenched AA-induced channel activation, indicating that the effect of intracellular AA on Na(V) was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of I(Na) was mimicked by cAMP and quenched by a PKA (protein kinase A) inhibitor, a G(s) inhibitor and EP (E-series of prostaglandin) receptor antagonists. The results of the present study suggest that extracellular AA modulates Na(V) channel activity in rat cerebellar GCs without metabolic conversion, whereas intracellular AA augments the I(Na) by PGE(2)-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.

Brain natriuretic peptide modulates the delayed rectifier outward K(+) current and promotes the proliferation of mouse Schwann cells.

Brain natriuretic peptide (BNP) may act as a neuromodulator via its associated receptors (natriuretic peptide receptors, NPRs) in the central nervous system (CNS), but few studies have reported its activity in the peripheral nervous system (PNS). In this study, we observed that BNP increased the tetraethylammonium chloride (TEA)-sensitive delayed rectifier outward potassium current (I(K)) in mouse Schwann cells (SCs) using whole-cell recording techniques. At concentrations of 1-100 nM, BNP reversibly activated I(K) in a dose-dependent manner, with modulating its steady-state activation and inactivation properties. The effect of BNP on I(K) was abolished by preincubation with the specific antagonist of NPR-A, and could not be mimicked by application of NPR-C agonist. These results were supported by immunocytochemical findings indicating that NPR-A was expressed in SCs. The application of 8-Br-guanosine 3',5'-monophosphate (8-Br-cGMP) mimicked the effect of BNP on I(K), but BNP was unable to further increase I(K) after the application of cyclic guanosine monophosphate (cGMP). Genistein blocked I(K) and also completely eliminated the effects of BNP and cGMP on I(K). The selective K(V)2.1 subunit blocker, Jingzhaotoxin-III (JZTX-III), reduced I(K) amplitude by 30%, but did not abolish the increase effect of BNP on I(K) amplitude. In addition, BNP significantly stimulated SCs proliferation and this effect could be partly inhibited by TEA. Together these results suggest that BNP modulated I(K) probably via cGMP- and tyrosine kinase-dependent pathways by activation of NPR-A. This effect of BNP on I(K) in SCs might partly explain its effect on cell proliferation.

Modulation of muscle rNaV1.4 Na+ channel isoform by arachidonic acid and its non-metabolized analog.

Arachidonic acid (AA) and its metabolic products are important second messengers which exert many biological actions, including modulation of various ion channels. However, the blockage of muscle Na(+) channel isoforms by AA has not been examined in detail. Here, we investigated the modulating effects of AA on muscle rNa(V)1.4 isoforms expressed in human embryonic kidney 293 cells. The results revealed that AA has both activation and inhibitory effects on rNa(V)1.4 currents depending on the depolarizing potential: AA increased the rNa(V)1.4 current evoked by a depolarization of -30 or -40 mV, but significantly decreased the rNa(V)1.4 current evoked by a depolarization of membrane potential over -10 mV. At concentrations of 1-500 microM, the inhibitory effect on the rNa(V)1.4 current induced by AA was dose-dependent and reversible. In addition to modulating the amplitude of the rNa(V)1.4 current, AA significantly modulated the steady-state activation and inactivation properties of rNa(V)1.4 channels. Furthermore, treatment with AA resulted in a fairly slow recovery of the rNa(V)1.4 channel from inactivation; however, the inhibitory effect of AA was not changed by repetitive pulses or by changing frequency. The effect of AA on rNa(V)1.4 currents was completely mimicked by ETYA, the non-metabolized analog of AA. Our data demonstrated that AA, but not the metabolic products of AA, can voltage-dependent modulate rNa(V)1.4 currents.

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells.

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.

C(6)-ceramide inhibited Na(+) currents by intracellular Ca(2+) release in rat myoblasts.

Ceramides are novel second messengers that may mediate signaling leading to apoptosis and the regulation of cell cycle progression. Moreover, ceramide analogs have been reported to directly modulate K(+) and Ca(2+) channels in different cell types. In this report, the effect of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)) in cultured rat myoblasts was investigated using whole-cell current recording and a fluorescent Ca(2+) imaging experiment. At concentrations of 1-100 microM, ceramide produced a dose-independent and reversible inhibition of I(Na). Ceramide also significantly shifted the steady-state inactivation curve of I(Na) by 16 mV toward the hyperpolarizing potential, but did not alter the steady-state activation properties. C(2)-ceramide caused a similar inhibitory effect on I(Na) amplitude. However, dihydro-C(6)-ceramide, the inactive analog of ceramide, failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent BAPTA, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with xestospongin C or heparin, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, ruthenium red, which is a blocker of the ryanodine-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide on I(Na). Intracellular application of the G-protein agonist GTPgammaS also induced a gradual decrease in I(Na) amplitude, while the G-protein antagonist GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Calcium imaging showed that C(6)-ceramide could give rise to a significant elevation of intracellular calcium. Our data show that increased calcium release through the IP(3)-sensitive Ca(2+) receptor, which probably occurred through the G-protein and phospholipase C pathway, may be responsible for C(6)-ceramide-induced inhibition of the I(Na) of rat myoblasts.