We have designed an epifluorescence video microscope for simultaneous dual excitation of indo 1 (for [Ca²⁺]_i) at 350 nm and SNARF 1 (for pH_i) 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 pH_i 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 (pH_i). 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 pH_i. 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 pH_i 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.

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

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