Abstract
In cultured bovine adrenal chromaffin cells expressing Nav1.7 isoform of voltage-dependent Na+channels, treatment (≥6 h) with serum deprivation, PD98059, or U0126 increased cell surface [3H]saxitoxin ([3H]STX) binding by ∼58% (t1/2 = 12.5 h), with no change in the Kd value. Immunoblot analysis showed that either treatment attenuated constitutive phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 but not of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) 1 and JNK2. The increase of [3H]STX binding and the attenuated phosphorylation of ERK1 and ERK2 returned to the control nontreated levels after the addition of serum or the washout of PD98059- or U0126-treated cells. Simultaneous treatment of serum deprivation with PD98059 or U0126 did not produce an additional increasing effect on [3H]STX binding, compared with either treatment alone. In cells subjected to either treatment, veratridine-induced maximum 22Na+ influx was augmented by ∼47%, with no change in the EC50 value;Ptychodiscus brevis toxin-3 enhanced veratridine-induced22Na+ influx by 2-fold, as in nontreated cells. Serum deprivation, PD98059, or U0126 increased Na+ channel α- but not β1- subunit mRNA level by ∼50% between 3 and 24 h; cycloheximide, an inhibitor of protein synthesis, increased α-subunit mRNA level and nullified additional increasing effect of either treatment on α-subunit mRNA level. Either treatment prolonged half-life of α-subunit mRNA from 17.5 to ∼26.3 h without altering α-subunit gene transcription. Thus, constitutively phosphorylated/activated ERK destabilizes Na+ channel α-subunit mRNA via translational event, which negatively regulates steady-state level of α-subunit mRNA and cell surface expression of functional Na+ channels.
Footnotes
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This research was supported by a grant from the Ichiro Kanehara Foundation, and by a Grant-in-Aid for 21st century COE (Centers of Excellence) Program (Life Science) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
- Abbreviations:
- DRG
- dorsal root ganglion
- APP
- amyloid precursor protein
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester
- DMSO
- dimethyl sulfoxide
- ERK
- extracellular signal-regulated kinase
- FRAP
- FK506 binding protein- and rapamycin-associated protein
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- hNE-Na
- human neuroendocrine type Na+channel α-subunit
- JNK
- c-Jun N-terminal kinase
- kb
- kilobase(s)
- KRP
- Krebs-Ringer phosphate
- MAPK
- mitogen-activated protein kinases
- MEK
- MAPK/ERK kinase
- nt
- nucleotides
- p38
- p38 mitogen-activated protein kinase
- pBII
- plasmid Bluescript II
- PbTx-3
- Ptychodiscus brevis toxin-3
- PKC
- protein kinase C
- RTK
- receptor tyrosine kinase
- SSC
- saline-sodium citrate
- STX
- saxitoxin
- TTX
- tetrodotoxin
- PD98059
- 2′-amino-3′-methoxyflavone
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole
- SP600125
- anthra[19-cd]pyrazol-6(2H)-one
- U0126
- 14-diamino-23-dicyano-14-bis(2-aminophenylthio)butadiene
- A23187
- calcimycin
- Received August 7, 2002.
- Accepted January 23, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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