Intracellular Compartmentalization of Fura-2 Dye Demonstrated by Laser-Excitation Fluorescence Microscopy: A Problem in Measuring Cytosolic Free Calcium Concentration Using Fura-2 Fluorescence in Vascular Smooth Muscle Cells

Kazuhisa Takeuchi, Kaoru Yoshinaga, Shun ichi Sato, Masato Kimura, Humio Inaba, Keishi Abe, Taka aki Abe

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

Takeuchi, K., Sato, S., Abe, K., Kimura, M., Abe, T., Yoshinaga, K. and Inaba, H. Intracellular Compartmentalization of Fura-2 Dye Demonstrated by Laser-Excitation Fluorescence Microscopy: A Problem in Measuring Cytosolic Free Calcium Concentration Using Fura-2 Fluorescence in Vascular Smooth Muscle Cells. Tohoku J. Exp. Med., 1989, 159 (1), 23-35 - Recently, we have developed a novel laser-excitation fluorescence microscope system to study intracellular calcium (Ca2+) in individual cultured vascular smooth muscle (VSM) cells using fluorescent indicators for Ca2+ In the cource of our study, it was shown that the subcellular fluorescence distribution of fura-2 was not homogeneous in VSM cells incubated with the acetoxy-methyl ester form of fura-2, fura-2/AM. The fluorescence appeared spotty or filamentous and resembled in shapes the intracellular organelles, suggesting that there was fura-2 dye compartmentalization in the organelles. To clarify the nature of the subcellular fluorescence, the soluble fraction of cells loaded with the dye was analyzed through high performance liquid chromatography (HPLC). We also examined the excitation spectra of fluorescence in the soluble fraction, which was compared with that in the cell suspension. Using HPLC, it has been shown that no other than fura-2 was found in the soluble fraction, whereas analyses of excitation spectra have indicated that the membrane fraction contained fura-2/AM or its lipophilic metabolite. On the other hand, indo-1 dye fluorescence showed a diffuse intracellular distribution, but the nuclear region had higher or sometimes lower fluorescence levels than the cytoplasm. The present results suggest that it may be necessary to assess subcellular fura-2 compartmentalization and possible interference by fura-2/AM or its lipophilic metabolite for the accurate measurement of intracellular Ca2+ concentration in VSM cells. It is also suggested that indo-1 may be more suitable for estimating Ca2+ concentration than fura-2 in individual VSM cells.

Original languageEnglish
Pages (from-to)23-35
Number of pages13
Journalthe tohoku journal of experimental medicine
Volume159
Issue number1
DOIs
Publication statusPublished - 1989
Externally publishedYes

Keywords

  • cytosolic free calcium
  • fura-2
  • indo-1
  • laserexcitation fluorescence microscope
  • smooth muscle cells

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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