Direct measurement of nitrite transport across erythrocyte membrane vesicles using the fluorescent probe, 6-methoxy-N-(3-sulfopropyl) quinolinium.
Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO2 in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO2- by a heteroexchange mechanism with other monovalent anions.
Shingles
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Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
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McCarty
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D000838:Anions; D001692:Biological Transport; D002462:Cell Membrane; D004910:Erythrocyte Membrane; D004912:Erythrocytes / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D009573:Nitrites / Q000378:metabolism*; D011522:Protons; D011806:Quinolinium Compounds
D000838:Anions; D005456:Fluorescent Dyes; D009573:Nitrites; D011522:Protons; D011806:Quinolinium Compounds; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium