title: Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells.
abstract: BACKGROUND
  Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown.
  
  
  RESULTS
  In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 μmol/l) ICl,swell and inhibited (IC50 = 6.9 μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution.
  
  
  CONCLUSIONS
  Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.
authors.affiliation: aDepartments of Physiology and Medicine bDivision of Cardiology, University Health Network, University of Toronto, Toronto, Canada cChina-Japan Union Hospital, Jilin University, Changchun, China dDivision of Respirology eThe Li Ka Shing Knowledge Institute, St. Michael's Hospital,University of Toronto, Toronto, Canada fCedars-Sinai Heart Institute, Los Angeles, California, USA *Wenbin Liang and Lihong Huang contributed equally to this study. **Deceased.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Liang
authors.forename: Lihong
authors.initials: L
authors.lastname: Huang
authors.forename: Dongling
authors.initials: D
authors.lastname: Zhao
authors.forename: Jeff Z
authors.initials: JZ
authors.lastname: He
authors.forename: Parveen
authors.initials: P
authors.lastname: Sharma
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Gramolini
authors.forename: Michael E
authors.initials: ME
authors.lastname: Ward
authors.forename: Hee Cheol
authors.initials: HC
authors.lastname: Cho
authors.forename: Peter H
authors.initials: PH
authors.lastname: Backx
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D049108:Cell Enlargement; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006801:Humans; D006976:Hypertension, Pulmonary / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D007189:Indans / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats
chemical_list: C443139:4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-one-5-yl)oxybutyric acid; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D003517:Cyclopentanes; D007189:Indans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid