title: Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis.
abstract: Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl(-)D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.
authors.affiliation: Department of Biological Sciences, 1275 University Esplanade, Kent State University, Kent, OH 44242, USA.
authors.forename: Brandon A
authors.initials: BA
authors.lastname: Gibbons
authors.affiliation: Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA.
authors.forename: Prakash
authors.initials: P
authors.lastname: Kharel
authors.affiliation: Department of Biological Sciences, 1275 University Esplanade, Kent State University, Kent, OH 44242, USA.
authors.forename: Lauren C
authors.initials: LC
authors.lastname: Robinson
authors.affiliation: J.A. Woollam Co., Inc., 645 M Street, Suite 102, Lincoln, NE 68508, USA. Electronic address: rsynowicki@jawoollam.com.
authors.forename: Ron A
authors.initials: RA
authors.lastname: Synowicki
authors.affiliation: Department of Biological Sciences, 1275 University Esplanade, Kent State University, Kent, OH 44242, USA. Electronic address: mmodel@kent.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Model
mesh_terms: D017209:Apoptosis; D002463:Cell Membrane Permeability; D048430:Cell Shape; D048429:Cell Size; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D027701:Organic Cation Transport Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D020112:Rhodamine 123 / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D027701:Organic Cation Transport Proteins; D020112:Rhodamine 123; D012964:Sodium
keywords: Apoptosis; Blebbing; Cell volume; Organic cation transport; Transmission-through-dye microscopy