Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

Abstract

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1–2 s) trains of stimuli. With fluorescence excitation at either 350 ± 20 or 450 ± 50 nm, and with emission measured, respectively, at 450 ± 50 or ≥ 520 nm, ΔF/F _o was ∼5–8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by ∼100 ms and were eliminated by 1 μM tetrodotoxin (TTX). The signals were partially inhibited by 500 μM Cd²⁺, by substitution of Mg²⁺ for Ca²⁺, by Ca²⁺-free Ringer’s with 0.5 mM EGTA, and by 50 μM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 μM), FCCP (0.3 μM), and NaN₃ (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO₂) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH₂ is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca²⁺]_i triggers an increase in FAD and NAD as FADH₂ and NADH are oxidized, but that elevation of [Ca²⁺]_i, alone cannot account for the totality of changes in intrinsic fluorescence.