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Am J Physiol Cell Physiol 276: C747-C757, 1999;
0363-6143/99 $5.00
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Vol. 276, Issue 3, C747-C757, March 1999

SPECIAL COMMUNICATION
Synthesis and characterization of dual-wavelength Clminus -sensitive fluorescent indicators for ratio imaging

Sujatha Jayaraman, Joachim Biwersi, and A. S. Verkman

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521

The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavelength (lambda ex) 350 nm, emission wavelength (lambda em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl--insensitive moiety because of its different spectral characteristics (lambda ex 380 nm, lambda em 546 nm), insensitivity to Cl-, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl--sensitive indicators were alpha -MQ-alpha '-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-alpha '-MQ-1'-alpha '-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer constants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes in Cl- concentration in response to Cl-/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl- indicators for ratiometric measurement of Cl- concentration.

6-methoxy-N-3-(sulfopropyl)quinolinium; fluorescence; chloride transport; membrane permeability; organic synthesis


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