Document Detail


Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein.
MedLine Citation:
PMID:  12645993     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Intracellular free H+ concentration (pHi) responds to numerous extracellular stimuli. The use of fluorescent indicator dyes to measure pHi is strongly influenced by the ability of target cells to retain activated dye within the cytoplasmic compartment. Here, 3 pH-sensitive indicator dye - acetoxymethyl (AM) esters of SNARF-1 and BCECF, and the thiol-reactive 5-chloromethyfluorescein (CMFDA) - were examined for monitoring pHi. The stability of pH measurements was strongly affected by temperature, cell type, indicator dye, and use of transport inhibitors to prevent dye export. Cellular retention of CMFDA, which forms covalent complexes, was sufficient to permit monitoring of transient pHi changes over extended time periods in a multi-well plate assay format. In human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells, increasing osmotic pressure caused a significant rise in pHi. In contrast, activation of native or transfected beta-adrenergic, cholinergic, and d and m opioid receptors did not measurably affect pHi in HEK293 cells. Decreases in pHi were observed in CHO cells expressing the human H+/peptide transporter PEPT1 upon addition of dipeptide substrates. The use of CMFDA in multi-well formats should facilitate study of osmotic and transport activity and screening for drugs that affect pHi.
Authors:
Aline Salvi; J Mark Quillan; Wolfgang Sadée
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  AAPS pharmSci     Volume:  4     ISSN:  1522-1059     ISO Abbreviation:  AAPS PharmSci     Publication Date:  2002  
Date Detail:
Created Date:  2003-03-20     Completed Date:  2003-03-31     Revised Date:  2013-06-09    
Medline Journal Info:
Nlm Unique ID:  100897065     Medline TA:  AAPS PharmSci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  E21     Citation Subset:  IM    
Affiliation:
Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0446, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Transport
CHO Cells
Cell Membrane
Cells, Cultured
Cricetinae
Fluoresceins / metabolism*
Fluorescent Dyes / metabolism*
Humans
Hydrogen-Ion Concentration
Osmotic Pressure
Grant Support
ID/Acronym/Agency:
GM43102/GM/NIGMS NIH HHS; GM61390/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Fluoresceins; 0/Fluorescent Dyes; 136832-63-8/5-chloromethylfluorescein
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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