By Ting-TingZhang, KoichiTakimoto, Alexandre F. R.Stewart, ChongxueZhu and Edwin S.Levitan
Ang II and PE induce hypertrophic responses. Rat neonatal cardiac myocytes were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or...Show More
Ang II and PE induce hypertrophic responses. Rat neonatal cardiac myocytes were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or 24 hours. Medium containing the agent or vehicle was changed every 12 hours. Amount of β-MHC mRNA was measured with RNase protection assay and normalized with an internal control β-actin mRNA (n≥3).Show Less
By Ting-TingZhang, KoichiTakimoto, Alexandre F. R.Stewart, ChongxueZhu and Edwin S.Levitan
Ang II and PE decrease Kv4.3 mRNA and protein expression. A, Four days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or...Show More
Ang II and PE decrease Kv4.3 mRNA and protein expression. A, Four days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 8 or 24 hours. Medium containing the agent or vehicle was changed every 12 hours. Myocytes were harvested, and Kv4.3 mRNAs were measured using RNase protection assay. In the upper 2 panels, β-actin mRNA was used as an internal control. Bottom panel shows results from an experiment with 8-hour treatments in which 28S ribosomal RNA was used as an internal control. P indicates probe; Y, yeast RNA; and C, control. B, Time courses of Ang II–induced and PE-induced Kv4.3 mRNA downregulation (n≥3 at each time point, normalization with β-actin). C, Three days after plating, cells were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicle for 60 hours. Medium containing the agent or vehicle was changed every 24 hours. Total protein was isolated, and Kv4.3 proteins were detected using an anti-Kv4.3 antibody.Show Less
An AT1 receptor antagonist inhibits the Ang II–induced decrease in Kv4.3 mRNA but does not affect the function of PE. Myocytes were pretreated with the AT1 receptor blocker L158,809 (0.5 μg/mL in DMSO, investigational drug from Merck) for 30 minutes. A, Ang II (100 nmol/L) or vehicle were then added in the continuing presence of L158,809 for 8 hours. Kv4.3 mRNA levels were measured using RNase protection assay (n=3). B, PE (100 μmol/L) or vehicle were added into the plate containing L158,809 for 8 or 24 hours. Kv4.3 mRNA levels were measured using RNase protection assay (n=3).
By Ting-TingZhang, KoichiTakimoto, Alexandre F. R.Stewart, ChongxueZhu and Edwin S.Levitan
Effects of Ang II and PE on Kv4.3 gene expression are additive. Cells were treated with vehicle, Ang II (100 nmol/L), PE (100 μmol/L), or both hormone...Show More
Effects of Ang II and PE on Kv4.3 gene expression are additive. Cells were treated with vehicle, Ang II (100 nmol/L), PE (100 μmol/L), or both hormones for 8 or 24 hours. Medium containing the agent or vehicle was changed every 12 hours. Total RNAs were isolated, and Kv4.3 mRNA levels were measured (n=3).Show Less
PE does not affect Kv4.3 mRNA stability. Four days after plating, cells were incubated with the transcription inhibitor actinomycin D (2 μmol/L) for various times. Kv4.3 mRNA levels were measured using RNase protection assays. α1B mRNA was measured as a positive control. GAPDH mRNA was for internal normalization. A, Top and bottom panels show adrenergic receptor α1B and Kv4.3 mRNA levels, respectively. B, Time courses of changes in adrenergic receptor α1B and Kv4.3 mRNAs with the actinomycin D treatment (n=3). C, Actinomycin D was added after pretreatment with PE (100 μmol/L) or vehicle for 24 hours. Kv4.3 mRNA levels at indicated incubation time with actinomycin D are shown (n=3).
The 5′ flanking region of the rat Kv4.3 gene and transcription start sites. A, RNase protection assays with adult rat brain (20 μg), heart (30 μg), and rat neonatal myocyte (20 μg) RNAs were performed. RNA probe corresponded to a fragment encompassing from 362 bp upstream to 326 bp downstream of the translation initiation site. The size of protected fragments was estimated using [32P]end-labeled marker (PhiX174 DNA/Hinf1 dephosphorylated marker, Promega). B, 5′ RACE analysis of adult rat brain and rat neonatal myocyte mRNAs was performed as described in Materials and Methods. C, Sequence of Kv4.3 5′ flanking region and potential transcription factor binding motifs. The determined transcription start sites of Kv4.3 mRNA in cardiac myocytes and brain are indicated by arrows. Bold ATG indicates the translation initiation site. AP1, nuclear factor-κB, and GATA potential binding motifs are underlined. Restriction enzyme sites of NcoI and XhoI are marked.
Effects of Ang II and PE on Kv4.3 promoter activity in neonatal myocytes. A, Neonatal myocytes were transfected with pGL3-luciferase basic vector inserted with various lengths of Kv4.3 fragments upstream of the coding region (BamHI, BglII, PvuII, and XhoI to NcoI, corresponding to 2337, 1157, 663, and 295 bp to 2 bp upstream of Kv4.3 translation start site) or vector alone. Luciferase activities were measured and normalized with an internal Renilla luciferase control driven by the herpes simplex virus thymidine kinase promoter. The luciferase activity obtained with the vector alone is set at one. Luciferase activities in cells transfected with 2337-bp and 1157-bp constructs are averages of 2 experiments. Luciferase activities of the other 2 constructs are averages of 5 experiments. B, Forty hours after transfection with the smallest construct (XhoI to NcoI), myocytes were treated with Ang II (100 nmol/L), PE (100 μmol/L), or vehicles for 7 hours. Luciferase activities were then measured (n≥5).
