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Proceedings Paper

Simultaneous multiple wavelength fluorescence video microscopy shows Ca2+ regulation of pH in living cells
Author(s): Stephen J. Morris; Diane M. Beatty; Bibie M. Chronwall
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Paper Abstract

We have designed an epifluorescence video microscope for simultaneous dual excitation of indo 1 (for [Ca2+]i) at 350 nm and SNARF 1 (for pHi) at 540 nm. The microscope will simultaneously capture all four emission images at 405, 475, 575, and 640 nm from the two ratio dyes at video frame or field rates. Popular dyes for measuring [Ca2+]i, such as indo 1 and fura 2, have pH- dependent Kd's; thus changes in pHi can be misinterpreted as changes in [Ca2+]i. For any pixel (or region of interest), we use the pH value to generate the appropriate Kd. The corrected Kd is then used to calculate a corrected calcium value. Using the imaging system, we show that, for the peptide secreting melanotropes of the pituitary intermediate lobe, changes in intracellular calcium ([Ca2PLU)]i) produce changes in intracellular pH (pHi). Melanotropes grown in primary explant culture and double-loaded with indo-1 acetoxy methyl ester (AM) and SNARF-1 AM, were examined for Ca2+/pH interactions. Following experimentation, cells were positively identified by (beta) -endorphin fluorescence immunohistochemistry. K-induced depolarization of melanotropes produced increases in [Ca2PLU)]i due to activation of L-type Ca-channels. Ca2+ entry was closely coupled to reductions in pHi. Effects were dependent upon entry of extracellular Ca2+ rather than release from intracellular stores. The close association between increases in intracellular Ca2+ and H+ suggest that the pHi changes are due to release of H+ upon binding of Ca2+ to intracellular buffers. Although the pH drop is `passive,' it will be sensed by all cytoplasmic components. Thus it represents a second messenger pathway, akin to the generation of cAMP or inositol trisphosphate, which cannot fail to influence numerous pH-dependent cell activities.

Paper Details

Date Published: 17 August 1994
PDF: 12 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182714
Show Author Affiliations
Stephen J. Morris, Univ. of Missouri/Kansas City (United States)
Diane M. Beatty, Univ. of Missouri/Kansas City (United States)
Bibie M. Chronwall, Univ. of Missouri/Kansas City (United States)


Published in SPIE Proceedings Vol. 2137:
Time-Resolved Laser Spectroscopy in Biochemistry IV
Joseph R. Lakowicz, Editor(s)

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