Monochloramine impairs caspase-3 through thiol oxidation and Zn2+ release.
BACKGROUND
Caspase-3, a pro-apoptotic enzyme, represents a class of proteins in which the active site contains reduced thiol (S-H) groups and is modulated by heavy metal cations, such as Zn(2+). We explored the effects of the thiol oxidant monochloramine (NH(2)Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH(2)Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn(2+) within the cytoplasm.


METHODS
Isolated gastric glands were prepared from rabbit mucosa by collagenase digestion. Caspase-3 activity was measured colorimetrically in suspensions of healthy rabbit gastric glands, following exposure to various concentrations of NH(2)Cl with or without the zinc chelator TPEN [tetrakis-(2-pyridylmethyl)ethylene diamine] for 1 h, and re-equilibration in Ringer's solution for 5 h. Conversion of procaspase-3 to active caspase-3 was monitored by Western blot.


RESULTS
Monochloramine inhibited caspase-3 activity in a dose-dependent fashion. At concentrations of NH(2)Cl up to 100 microM, these effects were prevented if TPEN was given concurrently and were partly reversed if TPEN was given 1 h later. Caspase-3 activity was preserved by concurrent treatment with a thiol-reducing agent, dithiothreitol.


CONCLUSIONS
At pathologically relevant concentrations, NH(2)Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH(2)Cl-induced accumulation of Zn(2+) in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH(2)Cl, impair host pathways of apoptosis through release of Zn(2+) from endogenous pools.
Kohler
Jonathan E
JE
Department of Surgery, Brigham & Women's Hospital, Boston, MA 02115, USA.
Mathew
Jeff
J
Tai
Kaniza
K
Blass
Amy L
AL
Kelly
Edward
E
Soybel
David I
DI
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D002700:Chloramines / Q000494:pharmacology*; D004195:Disease Models, Animal; D005753:Gastric Mucosa / Q000187:drug effects*; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D002700:Chloramines; D016877:Oxidants; D013438:Sulfhydryl Compounds; D053148:Caspase 3; D015032:Zinc; C030816:chloramine