Neuronal acid-induced [Zn²⁺]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.
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 [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 μM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 μM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]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 [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines.
The Psychiatric Institute, Departments of Psychiatry and Pharmacology, The University of Illinois at Chicago, Chicago, Illinois, USA.
Lech
L
Kiedrowski
D000143:Acids / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005029:Ethylenediamines; D005033:Ethylmaleimide / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D011083:Polycyclic Compounds; D061209:Proton Ionophores / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D000143:Acids; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005029:Ethylenediamines; C451182:FluoZin-3; D011083:Polycyclic Compounds; D061209:Proton Ionophores; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium
ATP; Acidification; FCCP; FluoZin-3; NEM; Zinc