Yonsei Med J.  2012 Jul;53(4):842-848. 10.3349/ymj.2012.53.4.842.

Fluoxetine Protects against Big Endothelin-1 Induced Anti-Apoptosis by Rescuing Kv1.5 Channels in Human Pulmonary Arterial Smooth Muscle Cells

• Dai F
• Mao Z
• Xia J
• Zhu S
• Wu Z

• KMID: 1716887
• DOI: http://doi.org/10.3349/ymj.2012.53.4.842

Abstract

PURPOSE
Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro.
MATERIALS AND METHODS
Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected.
RESULTS
In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis.
CONCLUSION
The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca2+.

Keyword

Apoptosis; Kv1.5; human pulmonary arterial smooth muscle cells

MeSH Terms

Apoptosis/drug effects/genetics
Blotting, Western
Cell Proliferation/drug effects
Cells, Cultured
Endothelin-1/*pharmacology
Flow Cytometry
Fluoxetine/*pharmacology
Humans
Kv1.5 Potassium Channel/genetics/*metabolism
Muscle, Smooth, Vascular/*cytology/drug effects
Pulmonary Artery/*cytology
Reverse Transcriptase Polymerase Chain Reaction

Figure

• 

  Fig. 1 Fluoxetine mediated anti-proliferation of human PASMCs against ET-1. (A) Time course of the 2% FBS (Blank) and ET-1 (0.01 to 1 µM) induced human PASMCs proliferation (B). (C) The anti-proliferation of fluoxetine in a dose-dependent manner against ET-1 (0.1 µM). (D) The approximate proliferation between the 2% FBS and fluoxetine (1.0 µM) treated groups, induced by ET-1 (0.1 µM). PASMCs, pulmonary arterial smooth muscle cells; FBS, fetal bovine serum; ET-1, endothelin-1; MTT, multiply-table tournament.

• 

  Fig. 2 Fluoxetine down regulated the expression of TRPC1 protein in ET-1 induced human PASMCs. Western blot results are displayed for TRPC1 (92 kDa) and GAPDH (34 kDa) in the human PASMCs cultured with low-serum DMEM (2% FBS, Blank), ET-1, and fluoxetine with (ET-1+F) and without ET-1 (F) for 72 hours. Data, normalized to the amount of actin, are expressed as mean±SEM (n=7). *p<0.01 vs. Blank, †p<0.05 vs. ET-1. PASMCs, pulmonary arterial smooth muscle cells; FBS, fetal bovine serum; TRPC, transient receptor potential channels; ET-1, endothelin-1; GAPDH, glyceraldehydes phosphate dehydrogenase; SEM, standard error of mean.

• 

  Fig. 3 Fluoxetine rescued the expression of Kv1.5 mRNA that was down regulated by ET-1 in human PASMCs. PCR amplified products are displayed for Kv1.5 (306 bp) and GAPDH (232 bp) in the human PASMCs cultured with low-serum DMEM (2% FBS, Blank), ET-1, and fluoxetine with (ET-1+F) and without ET-1(F) for 72 hours. Data, normalized to the amount of GAPDH, are expressed as mean±SEM (n=7). *p<0.05 vs. Blank, †p<0.001 vs. ET-1, ‡p<0.001 vs. Blank. PASMCs, pulmonary arterial smooth muscle cells; ET-1, endothelin-1; GAPDH, glyceraldehydes phosphate dehydrogenase; DMEM, dulbecco's modified eagle medium; SEM, standard error of mean; PCR, polymerase chain reaction.

• 

  Fig. 4 Fluoxetine reversed the level of Kv1.5 protein in ET-1 induced human PASMCs. Western blot results are displayed for Kv1.5 (57-59 kDa) and GAPDH (34 kDa) in the human PASMCs cultured with low-serum DMEM (2% FBS, Blank), ET-1, and fluoxetine with (ET-1+F) and without ET-1(F) for 72 h. Data, normalized to the amount of actin, are expressed as mean±SEM (n=9). *p<0.001 vs. Blank, †p<0.001 vs. ET-1. PASMCs, pulmonary arterial smooth muscle cells; FBS, fetal bovine serum; ET-1, endothelin-1; GAPDH, glyceraldehydes phosphate dehydrogenase; DMEM, dulbecco's modified eagle medium; SEM, standard error of mean.

• 

  Fig. 5 Fluoxetine enhanced the apoptosis ratio in ET-1induced human PASMCs. The result from Flow Cytometry are displayed for the ratio of apoptosis in the human PASMCs cultured with low-serum DMEM (2% FBS, Blank), ET-1, and fluoxetine with (ET-1+F) and without ET-1(F) for 72 h. PASMCs, pulmonary artery smooth muscle cells; FBS, fetal bovine serum; ET-1, endothelin-1; DMEM, dulbecco's modified eagle medium; PI, Propidium Iodide. FITC, fluoresceine isothiocyanate.

• 

  Fig. 6 Fluoxetine enhanced the apoptosis ratio in ET-1induced human PASMCs. The result from Flow Cytometry are displayed for the ratio of apoptosis in the human PASMCs cultured with low-serum DMEM (2% FBS, Blank), ET-1, and fluoxetine with (ET-1+F) and without ET-1(F) for 72 h. Data are expressed as mean±SEM (n=6). *p<0.01 vs. Blank, †p<0.001 vs. ET-1. PASMCs, pulmonary artery smooth muscle cells; FBS, fetal bovine serum; ET-1, endothelin-1; DMEM, dulbecco's modified eagle medium; SEM, standard error of mean.

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