Takayuki Nemoto

Lithium inhibits function of voltage-dependent sodium channels and catecholamine secretion independent of glycogen synthase kinase-3 in adrenal chromaffin cells

Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na(+) channels, nicotinic acetylcholine receptors, and voltage-dependent Ca(2+) channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced (22)Na(+) influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced (22)Na(+) influx, as well as inhibitory effect of LiCl on veratridine-induced (22)Na(+) influx. Enhancement of veratridine (site 2 toxin)-induced (22)Na(+) influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced (45)Ca(2+) influx and catecholamine secretion. In contrast, LiCl (< or = 30 mM) had no effect on nicotine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion, as well as on high K(+)-induced (45)Ca(2+) influx and catecholamine secretion. Chronic treatment with LiCl at 100mM (but not at < or = 30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na(+) influx thorough Na(+) channels and subsequent Ca(2+) influx and catecholamine secretion, independent of GSK-3 inhibition.

Regulation of Akt mRNA and protein levels by glycogen synthase kinase-3β in adrenal chromaffin cells: Effects of LiCl and SB216763

In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.

Up-regulation of NaV1.7 sodium channels expression by tumor necrosis factor-α in cultured bovine adrenal chromaffin cells and rat dorsal root ganglion neurons.

BACKGROUND:Tumor necrosis factor-&agr; (TNF-&agr;) is not only a key regulator of inflammatory response but also an important pain modulator. TNF-&agr; enhances both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant Na+ channel currents in dorsal root ganglion (DRG) neurons. However, it remains unknown whether TNF-&agr; affects the function and expression of the TTX-S NaV1.7 Na+ channel, which plays crucial roles in pain generation. METHODS:We used cultured bovine adrenal chromaffin cells expressing the NaV1.7 Na+ channel isoform and compared them with cultured rat DRG neurons. The expression of TNF receptor 1 and 2 (TNFR1 and TNFR2) in adrenal chromaffin cells was studied by Semiquantitative reverse transcription-polymerase chain reaction. The effects of TNF-&agr; on the expression of NaV1.7 were examined with reverse transcription-polymerase chain reaction and Western blot analysis. Results were expressed as mean ± SEM. RESULTS:TNFR1 and TNFR2 were expressed in adrenal chromaffin cells, as well as reported in DRG neurons. TNF-&agr; up-regulated NaV1.7 mRNA by 132% ± 9% (N = 5, P = 0.004) in adrenal chromaffin cells, as well as 117% ± 2% (N = 5, P < 0.0001) in DRG neurons. Western blot analysis showed that TNF-&agr; increased NaV1.7 protein up to 166% ± 24% (N = 5, corrected P < 0.0001) in adrenal chromaffin cells, concentration- and time-dependently. CONCLUSIONS:TNF-&agr; up-regulated NaV1.7 mRNA in both adrenal chromaffin cells and DRG neurons. In addition, TNF-&agr; up-regulated the protein expression of the TTX-S NaV1.7 channel in adrenal chromaffin cells. Our findings may contribute to understanding the peripheral nociceptive mechanism of TNF-&agr;.

Chronic lithium treatment up-regulates cell surface NaV1.7 sodium channels via inhibition of glycogen synthase kinase-3 in adrenal chromaffin cells: Enhancement of Na+ influx, Ca2+ influx and catecholamine secretion after lithium withdrawal

In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM, > or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.

Constitutive activity of glycogen synthase kinase-3β : Positive regulation of steady-state levels of insulin receptor substrates-1 and -2 in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, 12-h treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser(9) phosphorylation of GSK-3beta by approximately 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by approximately 38 and approximately 62% in a concentration-dependent manner. Treatment with SB216763 (0.1-30 microM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by approximately 38 and approximately 48%, while increasing beta-catenin protein level by approximately 47%, due to the prevention of GSK-3-induced degradation of beta-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser(9) phosphorylation of GSK-3beta by approximately 104%, while decreasing IRS-1 and IRS-2 levels by approximately 41 and approximately 72%; the insulin-induced Ser(9) phosphorylation of GSK-3beta, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin beta-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by approximately 100 and approximately 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3beta in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.

Insulin-induced neurite-like process outgrowth: Acceleration of tau protein synthesis via a phosphoinositide 3-kinase∼mammalian target of rapamycin pathway

Both insulin and tau, promoting neuronal differentiation (neurite outgrowth, neuronal polarity, and myelination) and cell survival, are associated with neurodegenerative disease (e.g., Alzheimers disease). The aim of this study was to explore relation between insulin-induced activation of insulin signal and expression of tau protein on neurite-like process outgrowth in adrenal chromaffin cells. Primary cultured bovine adrenal chromaffin cells were incubated with insulin to determine whether stimulant of insulin signal could affect tau expression and neurite-like process outgrowth. Chronic treatment with insulin (⩾6h) led neurite-like process outgrowth as well as increased tau protein level by ∼99% in a concentration (EC(50) 5.5nM)- and time-dependent manner, without changing Ser(396)-phosphorylated tau level. The insulin-induced increase of tau protein level was abolished by LY294002 [an inhibitor of phosphoinositide 3-kinase (PI3K)] and rapamycin [an inhibitor of mammalian target of rapamycin (mTOR)], but not by PD98059 and U0126 [two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)]. Additionally, insulin-induced increase of tau was blocked by cyclohexamide (an inhibitor of protein synthesis), but not by actinomycin D (an inhibitor of gene transcription). Pulse-label followed by polyacrylamide gel electrophoresis revealed that insulin accelerated tau protein synthesis rate (t(1/2)) from 2.6 to 1.9h. Insulin did not change tau mRNA level. Taken together, these results suggest that insulin-induced activation of PI3K∼mTOR pathway up-regulated tau protein via acceleration of protein synthesis, on which insulin promoted neurite-like process outgrowth.

Homologous posttranscriptional regulation of insulin-like growth factor-I receptor level via glycogen synthase kinase-3β and mammalian target of rapamycin in adrenal chromaffin cells: Effect on tau phosphorylation

In cultured bovine adrenal chromaffin cells, approximately 24 h-treatment with insulin-like growth factor-I (IGF-I) decreased cell surface (125)I-IGF-I binding capacity and IGF-I receptor protein level by approximately 64% (EC(50) = 5.0 nM; t(1/2) = approximately 7 h). IGF-I-induced IGF-I receptor decrease was abolished by LY294002 (phosphoinositide 3-kinase inhibitor) and partially attenuated by rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]). SB216763 (an inhibitor of glycogen synthase kinase-3 [GSK-3]) down-regulated IGF-I receptor, which was further decreased by IGF-I. IGF-I increased inhibitory Ser(9)-phosphorylation of GSK-3beta and stimulatory Ser(2448)-phosphorylation of mTOR. l-leucine increased phosphorylation of mTOR (but not GSK-3beta), and down-regulated IGF-I receptor, both events being abolished by rapamycin. IGF-I-induced IGF-I receptor decrease was not prevented by proteolysis inhibitors. Pulse-label with [(35)S]methionine/cysteine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that SB216763 or L-leucine retarded synthesis of IGF-I receptor and its precursor molecule. SB216763 (but not l-leucine) destabilized IGF-I receptor mRNA and decreased its level, without changing IGF-I receptor gene transcription. In SB216763-treated cells, IGF-I-induced Tyr-autophosphorylation of IGF-I receptor was decreased by 36%, compared to nontreated cells. IGF-I attenuated constitutive Ser(396)-phosphorylation of tau by 30% in nontreated cells, but not in SB216763-treated cells. IGF-I-induced down-regulations of (125)I-IGF-I binding and IGF-I receptor, as well as IGF-I-induced phosphorylations of GSK-3beta and mTOR were restored to the control levels of nontreated cells after washout of IGF-I (10 nM for 12 h)-treated cells. Thus, IGF-I down-regulated functional IGF-I receptor via GSK-3beta inhibition and mTOR activation; constitutive activity of GSK-3beta maintained IGF-I receptor level in nonstimulated cells.

Nav1.7 sodium channel-induced Ca2+ influx decreases tau phosphorylation via glycogen synthase kinase-3β in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells expressing Na(v)1.7 sodium channel isoform, veratridine increased Ser(473)-phosphorylation of Akt and Ser(9)-phosphorylation of glycogen synthase kinase-3beta by approximately 217 and approximately 195%, while decreasing Ser(396)-phosphorylation of tau by approximately 36% in a concentration (EC(50)=2.1 microM)- and time (t(1/2)=2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca(2+) removal. Veratridine (10 microM for 5 min) increased translocation of Ca(2+)-dependent conventional protein kinase C-alpha from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca(2+) removal, or Gö6976 (an inhibitor of protein kinase C-alpha), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3beta phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 microM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na(+) influx via Na(v)1.7 sodium channel and the subsequent Ca(2+) influx via voltage-dependent calcium channel activated (1) Ca(2+)/protein kinase C-alpha pathway, as well as (2) Ca(2+)/phosphatidylinositol 3-kinase/Akt and (3) Ca(2+)/phosphatidylinositol 3-kinase/protein kinase C-alpha pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation.

Proteasomal degradation of IRS-2, but not IRS-1 by calcineurin inhibition: Attenuation of insulin-like growth factor-I-induced GSK-3β and ERK pathways in adrenal chromaffin cells

The ability of calcineurin to regulate IRS-1 and IRS-2 levels has not been examined in any given cells, although calcineurin inhibition by therapeutic immunosuppressants produced cytoprotective and cytotoxic effects (e.g., new-onset of diabetes mellitus, seizure). Chronic (>or=3h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by approximately 50% (IC(50)=200 or 10nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3beta and ERK1/ERK2 protein levels. When the cells were washed to remove the test drug, the decreased IRS-2 level restored to the control level. Cyclosporin A or FK506 treatment inhibited calcineurin activity (IC(50)=500 or 40 nM, in vitro assay). Rapamycin, an FK506-binding protein ligand unable to inhibit calcineurin, failed to decrease IRS-2, but reversed FK506-induced decreases of calcineurin activity and IRS-2 level. Pulse-label followed by polyacrylamide gel electrophoresis revealed that cyclosporin A or FK506 accelerated IRS-2 degradation rate (t(1/2)) from >24 to approximately 4.2h, without altering IRS-2 synthesis. IRS-2 reduction by cyclosporin A or FK506 was prevented by lactacystin (proteasome inhibitor), but not by calpeptin (calpain inhibitor) or leupeptin (lysosome inhibitor). Cyclosporin A or FK506 increased serine-phosphorylation and ubiquitination of IRS-2. Cell surface (125)I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3beta and ERK1/ERK2 were attenuated by approximately 50%, which were prevented by rapamycin or lactacystin. Thus, calcineurin inhibition decreased IRS-2 level via proteasomal IRS-2 degradation, attenuating IGF-I-induced GSK-3beta and ERK pathways.

Dexmedetomidine and clonidine inhibit the function of NaV1.7 independent of α2-adrenoceptor in adrenal chromaffin cells

PurposeBesides being administered systemically for sedation and analgesia, α2-agonists such as dexmedetomidine and clonidine have been administered with intrathecal, epidural, or perineural injections, leading to an antinociceptive effect at the spinal cord or peripheral nerve level. However, the mechanism for this remains unclear. In the present study, we examined whether dexmedetomidine and clonidine could inhibit the function of tetrodotoxin-sensitive Na+ channels, which play important roles in the generation of pain.MethodsCultured bovine adrenal chromaffin cells expressing the tetrodotoxin-sensitive Nav1.7 Na+ channel isoform were incubated in KRP buffer containing 2xa0μCi 22NaCl for 5xa0min without or with dexmedetomidine or clonidine in the absence or presence of veratridine, α-scorpion venom, β-scorpion venom, Ptychodiscus brevis toxin-3 or ouabain. Cells were then washed and counted radioactively.ResultsDexmedetomidine and clonidine reduced veratridine-induced 22Na+ influx via Nav1.7 in a concentration-dependent manner (EC50xa0=xa050xa0μM and 530xa0μM), even in the presence of ouabain, an inhibitor of Na+, K+-ATPase. Dexmedetomidine and clonidine shifted the concentration–response curve of veratridine for 22Na+ influx downward without altering the EC50 of veratridine. Atipamezole and yohimbine, α2-antagonists, did not prevent the inhibition of veratridine-induced 22Na+ influx by dexmedetomidine. Dexmedetomidine and clonidine combined with lidocaine induced more inhibition of veratridine-induced 22Na+ influx than each drug did individually. Atipamezole and yohimbine did not prevent the lidocaine-enhancing effect of dexmedetomidine and clonidine.ConclusionDexmedetomidine and clonidine inhibit the function of Nav1.7 independent of α2-adrenoceptor. These results may lead to a deeper understanding of the peripheral antinociceptive effects of α2-agonists.