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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