کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
4012981 | 1261225 | 2006 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3â permeability of bovine corneal endothelium
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ایمونولوژی و میکروب شناسی (عمومی)
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چکیده انگلیسی
Corneal endothelium secretes HCO3â from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO3â permeability can be enhanced by increasing [Ca2+]i. We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca2+ activated apical HCO3â permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417-430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO3â permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90 kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca2+i] by application of ATPγS or cyclopiazonic acid (CPA) increased apical HCO3â permeability in siControl transfected CBCEC, while having no effect on apical HCO3â permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO3â permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca2+ dependent apical HCO3â permeability, but not resting permeability, across the corneal endothelium.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental Eye Research - Volume 83, Issue 5, November 2006, Pages 1215-1224
Journal: Experimental Eye Research - Volume 83, Issue 5, November 2006, Pages 1215-1224
نویسندگان
Yan Zhang, Jinhua Li, Qiang Xie, Joseph A. Bonanno,