Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes |
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Authors: | Masaru Watanabe Masato Konishi |
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Institution: | Department of Physiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. |
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Abstract: | Single ventricular myocytes enzymatically isolated from rat hearts were loaded with the Mg2+ indicator furaptra, and the relationship between the fluorescence ratio signal (R) and the intracellular free concentration of Mg2+ (Mg2+]i) was studied in situ at 25 degrees C. After the application of ionophores (ionomycin, monensin, nigericin and valinomycin), an immediate change in furaptra R was noted, followed by a slow change in R that reached a steady level in 2-4 h. The direction of the early change in R that accompanied rigor contraction was independent of the extracellular Mg2+ concentration (Mg2+]o), and was consistent with the breakdown of ATP and release of bound Mg2+. The intracellular calibration curve was constructed from the steady levels of R obtained at various Mg2+]o between 0 and 47 mM. The dissociation constant of intracellular furaptra was estimated to be 5.3 mM, which was 44% higher than that determined in salt solutions (3.7 mM). The basal Mg2+]i of rat ventricular myocytes calculated with the intracellular curve averaged 0.91 mM. |
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