Purchase this article with an account.
X. Sun, J.A. Bonanno; The Use of a Novel Membrane-Associated pHluorins for Measurements of Extracellular Surface pH in Bovine Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4714.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To measure the changes of extracellular surface pH in bovine corneal endothelial cells (BCEC) using a novel glycosylphosphatidylinositol (GPI)-anchored membrane-associated pH-sensitive green fluorescent protein-based sensor (pHluorin). Methods: Recombinant replicate-deficit adenovirus vectors were constructed with membrane (N-term) and GPI (C-term) signal peptide sequences linked to pHluorin. Cultured BCECs were exposed to virus (MOI=5) for 2 hours. At 24, 48 and 72 hours post infection, cells were observed by fluorescence ratio (FR, Ex 470/420) imaging. At 48 hours, membrane protein of transfected cells was tagged using NHS-biotin or cleaved using PI-PLC. Protein was subjected to western blot analysis using anti-GFP antisera. Results: Direct observation by standard epifluoresence microscopy showed that infection efficiency was 100% after 48 hours. Predominant GFP fluorescence was distributed in the cell membrane after 24 hours, but cytoplasmic fluorescence was observed in many cells, however the nucleus was devoid of fluorescence in all cells. Selecting cells with the minimum cytoplasmic fluorescence, we found that the FR changed by 20% in response to bath pH changes (6.5 to 7.5), but changed by <2% in response to NH4Cl pulse, indicating that the pH responsive signal is predominantly membrane associated. Western blot analysis confirmed that GFP was membrane associated and linked to a GPI stalk. Conclusions: This adenoviral vector can efficiently transfect primary cultures of endothelial cells and deliver the pHluorin to the extracellular membrane surface where it can detect changes in surface pH. Future experiments will be directed to measuring pH changes in response to transendothelial bicarbonate transport.
This PDF is available to Subscribers Only