Concurrent measurements of the free cytosolic concentrations of H+ and Na+ ions with fluorescent indicators.

Abstract

We report a method for the concurrent measurement of intracellular [Na+] ([Na+ ]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower p Ka in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.

• Authors: Claire Sheldon, Y May Cheng, John Church
• Journal: Pflugers Archiv : European journal of physiology (Pflugers Arch) Vol. 449 Issue 3 Pg. 307-18 (Dec 2004) ISSN: 0031-6768 [Print] Germany
• PMID: 15452716 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Benzofurans
• Benzopyrans
• Ethers, Cyclic
• Fluorescent Dyes
• Ions
• SNARF dye
• sodium-binding benzofuran isophthalate
• Hydrogen
• Sodium

Topics

• Animals
• Animals, Newborn
• Benzofurans
• Benzopyrans
• Cell Culture Techniques (methods)
• Ethers, Cyclic
• Fluorescent Dyes
• Hydrogen (metabolism)
• Hydrogen-Ion Concentration
• Intracellular Fluid (metabolism)
• Ions
• Microscopy, Fluorescence, Multiphoton (methods)
• Neurons (cytology, metabolism)
• Rats
• Rats, Wistar
• Sodium (metabolism)