Ca²⁺-Induced Deprotonation of Peptide Hormones Inside Secretory Vesicles in Preparation for Release

The depolarization-induced fluorescence change does not depend on exocytosis but requires extracellular Ca²⁺. Cells were pretreated with 0.2 mmNEM to block peptide release. A, Cells were superfused with normal saline and then with 100 mmK⁺ saline (bar). Note the apparent and uncontaminated increase in fluorescence. B, Cells were superfused with Ca²⁺-free normal saline, then with Ca²⁺-free 100 mmK⁺ saline, and finally with 100 mmK⁺ saline containing 5 mmCa²⁺. The fluorescence increase induced by 100 mm K⁺ saline occurred only when Ca²⁺ was present. C, Cd²⁺ prevented fluorescence increase caused by Ca²⁺. Cells were superfused with normal saline containing 0.2 mm Cd²⁺ and then with 100 mm K⁺ saline containing 0.2 mm Cd²⁺. D, Ba²⁺ mimicked Ca²⁺ in causing the fluorescence increase. Cells were initially superfused with normal saline and then with Ca²⁺-free Ba²⁺ containing 100 mmK⁺ saline. E, Quantification of divalent dependence of the effect of depolarization after NEM treatment. n = 4, 4, 6, and 4 for 0 Ca²⁺, 5 mm Ca²⁺, 5 mm Ca²⁺ plus 0.2 mmCd²⁺, and 5 mm Ba²⁺, respectively.

Fluorescence of EGFP-tagged peptide hormone is sensitive to pH. A, Fluorescence excitation spectra of proANF-EGFP fusion protein at two different pH values.B, Microfluorimetric recordings of the responses of proANF-EGFP fusion protein expressed in cells to solutions at various pH levels. The response of the fluorescence of proANF-EGFP to changing pH was quick and reversible. C, Titration curve for the relative fluorescence of proANF-EGFP.

The depolarization-induced increase in intravesicular fluorescence requires a pH gradient. Cells were initially superfused with normal saline containing a pH-collapsing agent [1 μm monensin (A), 1 μm nigericin (B), or 1 μm FCCP (C)] and then with 100 mm K⁺ saline containing the same pH-collapsing agent. Note that fluorescence decrease occurred almost immediately after 100 mmK⁺ application. D, Comparison of delay from the application of 100 mm K⁺to the onset of fluorescence decrease in cells without pretreatment (C) and those pretreated with monensin (M), nigericin (N), or FCCP (F). n = 11, 8, 5, and 5 for control, monensin, nigericin, and FCCP, respectively. *p < 0.01 versus control.

Fluorescence of proANF-Sapphire fusion protein is insensitive to pH. A, Fluorescence excitation spectra of proANF-Sapphire GFP fusion protein at two different pH values.B, Microfluorimetric recordings of the responses of proANF-Sapphire fusion protein to bath superfusion of normal saline at various pH levels. C, Titration curve for the relative fluorescence of Sapphire-tagged proANF.

The apparent delay after application of 100 mm K⁺ saline is no longer observed when using proANF-Sapphire. A, A cell was initially superfused with normal saline and then with 100 mmK⁺ saline. Note that fluorescence decrease occurred almost immediately after 100 mm K⁺application. Also, no increase in fluorescence was observed with proANF-Sapphire. B, Comparison of delay from the application of 100 mm K⁺ saline to the onset of fluorescence decrease in cells transfected with proANF-EGFP (EGFP) and those transfected with proANF-Sapphire (Sph). n = 11 and 12 for proANF-EGFP and proANF-Sapphire, respectively. *p < 0.01 versus EGFP.