Monochloramine impairs caspase-3 through thiol oxidation and Zn2+ release

Abstract

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.

Materials and 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.

Figure 1

Caspase-3 activity in isolated gastric glands in response to [Zn²⁺] in the presence of pyrithione (50μM) at increasing doses (2.5nM, 25nM, 250nM) for 3 hrs. Results are normalized to Ca²⁺ and Zn²⁺ depleted Ringer's controls (EGTA 0.5mM, pyrithione 50μM; average value 1.0) run concurrently and expressed as means ± SE, n=5 wells, *p<0.05 compared to Ringer's alone (ANOVA).

Figure 2

Caspase-3 activity in Isolated gastric glands in response to monochloramine at increasing doses (50 μM, 100 μM, 200 μM). Panel 2A: Exposure of glands to different concentrations of NH₂Cl for 2 hr, with immediate processing for caspase-3 activity. Panel 2B: Exposure of glands to NH₂CI at different doses for different periods of time (1hr, 2hr, 3 hr), after which glands were equilibrated with Ringer's for 5hr, 4hr, or 3hr, respectively, before processing. Each column represents 10 to 12 wells of glands from 3 to 4 separate preparations. Results are normalized to protein content (Bradford assay) and then to Ringer's controls (first white column in each group = 1.00) and expressed as means ± SE, *p<0.05 compared to Ringer's alone (ANOVA).

Figure 3

Caspase-3 activities in glands exposed to NH₂Cl at different concentrations, in the presence or absence of TPEN 20μM. Panel 3A: Glands were exposed to NH₂Cl alone (white bars) or TPEN (shaded bars) for 1 hr. Panel 3B: Glands were exposed to NH₂Cl for 1 hr, followed by equilibration in Ringer's, alone (white bars) or in the presence of TPEN (shaded bars). Each column represents 5-6 wells of glands from 3 separate preparations. Results are normalized to protein content (Bradford assay) and then to Ringer's controls (first white column in each group = 1.00) and expressed as means ± SE, *p<0.05 compared to Ringer's alone (ANOVA), ¶ indicates p<0.05 compared to effects without TPEN at the corresponding concentration.

Figure 4

Effects of delayed TPEN treatment after exposure to NH₂CI. Glands were perfused with Ringer's alone or Ringer's plus NH₂CI (100μM) for 1 1hr. Glands were perfused under similar conditions with TPEN present for the entire 6 hr incubation, or only for the 5 hr following exposure to NH₂CI. Each column represents 15 wells of glands from 3 separate preparations. Results are normalized to protein content (Bradford assay) and then to Ringer's controls (first white column in each group = 1.00) and expressed as means ± SE, *p<0.05 compared to Ringer's alone (ANOVA).

Figure 5

Comparison of effects of TPEN and DTT on inactivation of caspase-3 activity by NH₂CI. Glands were incubated for 2 hours at different concentrations of NH₂CI, alone or in the presence of the metal chelator TPEN or thiol reducing agent diothiothreitol (DTT). Results are normalized to protein content (Bradford assay) and then to Ringer's controls (first white column in each group = 1.00) and expressed as means ± SE, *p<0.05 compared to Ringer's or Ringer's plus NH₂Cl (ANOVA).