The halftime of the transient in cell chloride activity upon bath chloride addition was approximately 3 s (38 degrees C). Apparent chloride concentration in tubules perfused with solutions characteristic for the late proximal convoluted tubule was 27.5 +/- 5 mM (activity 20.6 mM). Cell fluorescence was related to intracellular chloride by a Stern-Volmer relation with a quenching constant of 12 M-1. Intracellular SPQ calibration was performed using the ionophores nigericin and tributyltin, high external potassium concentrations, and varying extracellular chloride concentrations. In suspended tubules, SPQ did not affect O2 consumption significantly. The halftime for SPQ leakage from cells at 38 degrees C was 8.6 +/- 1.1 min. The signal to background (autofluorescence) ratio was 4.6 +/- 0.6. Fluorescence was excited with a broad band excitation filter (340 and 380 nm) and detected with a 435 nm cut-on filter. SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38 degrees C. The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ).
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