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| CELL-PERMEABLE FLUORESCENT INDICATOR FOR CYTOSOLIC CHLORIDE |
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| Author(s): BIWERSI J, VERKMAN AS |
| Source: BIOCHEMISTRY Volume: 30 Issue: 32 Pages: 7879-7883 Published: AUG 13 1991 |
| Times Cited: 84 References: 26 |
| Abstract: A major limitation of quinolinium-based fluorescent indicators for cytosolic Cl- has been the necessity of invasive cell loading because the positively charged ring nitrogen confers high polarity and membrane impermeability. A novel approach to mask the positive nitrogen was developed and evaluated for rapid, noninvasive indicator loading into living cells and effective intracellular trapping. The nonpolar and lipophilic compound 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) was Cl- insensitive but was readily oxidized to the membrane-impermeable and Cl--sensitive fluorescent indicator 6-methoxy-N-ethylquinolinium chloride (MEQ). MEQ had 344-nm absorbance and 440-nm emission maxima, 0.70 quantum yield, and 4100 M-1 cm-1 molar extinction coefficient. In aqueous buffers, the fluorescence of MEQ was quenched by Cl- by a collisional mechanism with a Stern-Volmer constant (K(Cl)) of 145 M-1. MEQ fluorescence was quenched by other anions (K(Br) = 275 M-1, K(I) = 360 M-1, K(SCN) = 300 M-1) but not by NO3-, SO4(2-), cations, and pH. Swiss 3T3 fibroblasts and colonic T84 cells were loaded with MEQ by incubation at 37-degrees-C with 25-50-mu-M diH-MEQ for 5-10 min followed by diH-MEQ-free buffer for 15 min. MEQ stained cells brightly and uniformly and was nontoxic in studies of cell growth, cAMP and Ca2+ signaling, and electrophysiological properties. MEQ leaked out of cells by < 10% in 60 min and was sensitive to cytosolic Cl- with K(Cl) = 19 M-1. DiH-MEQ and similar reduced N-substituted dihydroquinolines should have applications in measurements of Cl- transport and regulatory mechanisms in living cells and epithelia. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV CALIF SAN FRANCISCO, CARDIOVASC RES INST, DEPT MED, 1065 HLTH SCI E TOWER, SAN FRANCISCO, CA 94143 USA
2. UNIV CALIF SAN FRANCISCO, CYST FIBROSIS RES CTR, SAN FRANCISCO, CA 94143 USA
3. UNIV CALIF SAN FRANCISCO, CARDIOVASC RES INST, DEPT PHYSIOL, SAN FRANCISCO, CA 94143 USA |
| Publisher: AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: GA940 |
| ISSN: 0006-2960 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |