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Use of the Cam Kinase Kinase β Inhibitor is Contraindicated in Calcium Imaging Studies
Author(s) -
Jurcsisn Jennifer,
Pye Richard Laurence,
Barr Barbara Lyn,
Wyatt Christopher Neil
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb798
Subject(s) - ionomycin , biophysics , ampk , kinase , chemistry , intracellular , calcium , protein kinase a , microbiology and biotechnology , biochemistry , biology , organic chemistry
The energy‐sensing enzyme AMP‐activated protein kinase (AMPK) may play a critical role in oxygen‐sensing by the carotid bodies. To activate AMPK, it must be phosphorylated at Thr‐172 (alpha subunit) by upstream kinases. This study's objective was to determine the importance of CamKKβ in the hypoxic chemotransduction mechanism of oxygen‐sensing Type I cells within the mouse carotid body. Type I cells were loaded with Fura‐2 (5 μM). Intracellular Ca 2+ was measured by exposing cells to 340nm and 380nm light and recording emission fluorescence at 510nm. Initial experiments were to see if Ca 2+ increase during hypoxic exposure (10 Torr) could be altered by the CamKKβ inhibitor STO609 (100 μM). However, STO609 is green in solution and application caused large emission signal deviations. Faster changes in the 380nm signal caused a 340/380 ratio fall, suggesting intracellular Ca 2+ decreased. Importantly, in the absence of cells, STO609 increased 510nm emission, indicating the inhibitor itself was fluorescing. To avoid this artifact, cells were loaded with a ‘red emission’ Ca 2+ indicator dye, x‐Rhod‐1 (5 μM), exposed to 580nm light, and emission was recorded at 602nm. Cells were excited with high K + (50mM). During elevated Ca 2+ , STO609 (100 μM) was applied and an immediate fall in the emission signal was observed. This decrease in Ca 2+ signal with STO609 was also seen in cells exposed to 0mM Ca 2+ solutions containing ionomycin. Thus STO609 directly interacts with x‐Rhod‐1 and is therefore precluded in Ca 2+ imaging studies. Supported by NIH RO1‐ HL091836