Neuronal acid‐induced [Zn²⁺]_i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

Abstract

The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn2+] ([Zn2+]i) elevations, a low affinity (Kd = 39 μM at pH 6.1) ratiometric Zn2+ probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn2+]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn2+]i increased further to 5.6 μM; analogous acid- and NEM-induced [Zn2+]i elevations could also be detected but not calibrated, using the high affinity Zn2+ probe FluoZin-3. The data indicate that NEM causes Zn2+ release from ligands that chelate Zn2+ at pH 6.1. ATP could also chelate Zn2+ at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn2+]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn2+]i increases. These data suggest that the total amount of Zn2+ accumulated in intracellular ATP-dependent stores (Zn2+-ATP complexes and organelles that accumulate Zn2+ in an ATP-dependent manner) is negligible compared to the amount of Zn2+ accumulated in the acid-sensitive intracellular ligands.

Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence

“…Our findings showed that cultured primary neurons from all three regions studied had similar total zinc contents (since each neuron had similar soma volumes) and showed a uniform subcellular distribution of zinc. This finding is consistent with the idea that the amount of free Zn 2+ accumulated in organelles (like that seen in the cytosol [ 27 , 29 ]) is negligible when compared with the amount coordinated via thiol and other groups and bound to proteins [ 19 , 29 ] giving zinc a fairly uniform average concentration throughout the cytoplasm and nucleus of individual somata.…”

“…To culture the neurons, neurobasal medium supplemented with 2% B27 and 2 mM glutamine was used. The cells were cultured in a humidified incubator maintained at 37°C and 5% O 2 , 5% CO 2 and 90% N 2 atmosphere [ 19 , 20 ].…”