The Effects of 1,10-Phenanthroline on the Binding of Activated Rat Hepatic Glucocorticoid-Receptor Complexes to Deoxyribonucleic Acid-Cellulose*

Abstract

1,10-Phenanthroline, a metal ion chelator, inhibits the binding of previously activated (25 C for 30 min) rat hepatic [3H]triamcinolone acetonide (3H-labeled 9-fluoro-11 beta, 21-dihydroxy-16 alpha, 17-1-[1-metylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione ([3H]TA)-receptor complexes to DNA-cellulose. The observed inhibition increases as the temperature of the preincubation with chelator is increased from 0 to 25 C. Fifty percent of the maximal inhibition (greater than 90%) detected at 25 C is achieved with 1 mM 1,10-phenanthroline. The observed inhibition is not the consequence of DNA degradation by 1,10-phenanthroline-Cu2+ complexes, since preincubation of activated cytosol with neocuproine (2,9-dimethyl-1,10-phenanthroline), a potent Cu2+ chelator, fails to block the subsequent inhibition of DNA-cellulose binding by 1,10-phenanthroline. The failure of other chelators which complex siilar metal ions (alpha, alpha'-dipyridyl,8-hydroxyquinoline, 2,2',2"-tripyridine, EDTA, EGTA, and Na azide) to inhibit DNA-cellulose binding suggests that the effectiveness of 1,10-phenanthroline does not result from removal of a required free metal ion(s) but, rather, from a specific interaction with a metal ion(s) which may be located within the activated receptor protein. The observed inhibition is dependent on the metal chelating properties of 1,10-phenanthroline, since preincubation with several divalent metal cations (Zn2+, Co2+, and Ni2+) which are known to be chelated by this compound block its subsequent inhibitory effect. Ferroin (1,10-phenanthroline-ferrous sulfate complex) and 1,7-phenanthroline (nonchelating isomer) also fail to inhibit DNA-cellulose binding. The inhibition mediated by 1,10-phenanthroline persists after gel filtration, suggesting that 1,10-phenanthroline associated with a macromolecule is the effective form of the inhibitor, rather than free 1,10-phenanthroline. Finally, 1,10-phenanthroline appears to interact directly with activated [3H]TA-receptor complexes, since it alters their net charge and results in their elution from DEAE-cellulose at a salt concentration characteristic of unactivated complexes. Collectively, the data suggest that the activated [3H]TA-receptor complex is a metalloprotein and that the metal ion(s) may be associated directly with the DNA-binding site or may regulate this site indirectly through an allostreic mechanism.

A role for metals and free radicals in the induction of apoptosis in thymocytes

“…One such attractive possibility is a metal and radical dependent transcriptional activation leading to apoptosis. It is interesting that 1, lo-phenanthroline blocks the binding of an activated glucocorticoid-receptor complex to DNA [53] and these authors suggested that the activated complex was a metalloprotein. Oxidative stress is known to activate the transcription factor NF-KB and antioxidants can inhibit this process [54,55].…”

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site

“…Calcium ions can also be dismissed since their free concentration was the same with and without EGTA in the dialysis solutions. In the case of H 2 O 2 , it could be argued that the Fenton reaction, which requires Fe 2ϩ , is involved (15,41 , and Ni 2ϩ are also unlikely to play any role because they are also chelated by phenanthroline (25,41,42,46). Consequently, the protection given by EGTA requires alternative explanations: either there are other participating cation/s with low affinity for phenanthroline and high affinity for EGTA, or perhaps EGTA, by binding to some sites of the exchanger protein, leads to conformational changes that reduce its sensitivity to oxidants.…”

“…In (25,41,42,45,46). Actually, phenanthroline has already been used in studies of redox modifications of Na ϩ /Ca 2ϩ exchange activity in mammalian heart sarcolemmal vesicles (41).…”

A new technique for staining mast cells using ferroin

“…Hukill and Putt (1962) employed a diphenylated derivative of o -phenanthroline (batho -phenanthroline) for detecting tissue iron. Ferroin has been used in several spectrophotometric studies and as a chelating agent of bivalent metals in different biological and chemical assays (Vydra and Kopanica 1963, Schmidt et al 1981, Allen et al 1983, Issopoulos 1992, Dunn et al 1993, Xu et al 1996, Boumans et al 1997, Luque-Pérez et al 2000. The use of ferroin for histochemical studies, however, has not been reported previously.…”