Amy L. Blass

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. 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 Ringers 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.

Self-assembled hydrogel fibers for sensing the multi-compartment intracellular milieu

Targeted delivery of drugs and sensors into cells is an attractive technology with both medical and scientific applications. Existing delivery vehicles are generally limited by the complexity of their design, dependence on active transport, and inability to function within cellular compartments. Here, we developed self-assembled nanofibrous hydrogel fibers using a biologically inert, low-molecular-weight amphiphile. Self-assembled nanofibrous hydrogels offer unique physical/mechanical properties and can easily be loaded with a diverse range of payloads. Unlike commercially available E. coli membrane particles covalently bound to the pH reporting dye pHrodo, pHrodo encapsulated in self-assembled hydrogel-fibers internalizes into macrophages at both physiologic (37°C) and sub-physiologic (4°C) temperatures through an energy-independent, passive process. Unlike dye alone or pHrodo complexed to E. coli, pHrodo-SAFs report pH in both the cytoplasm and phagosomes, as well the nucleus. This new class of materials should be useful for next-generation sensing of the intracellular milieu.

Redistribution of Labile Plasma Zinc During Mild Surgical Stress in the Rat

Zinc is an essential trace element and cofactor for many cellular processes. Uptake of ionized divalent zinc (Zn(2+)) in peripheral tissues depends on its total content in the circulation and on mechanisms facilitating delivery to tissues in its labile form. Understanding mechanisms of Zn(2+) delivery has been hindered by the absence of techniques to detect labile Zn(2+) in the circulation. In this study, we report the use of the fluorescent zinc-binding dye (ZnAF-2) to detect changes in labile Zn(2+) in the circulating plasma of the rat under standardized conditions, including exogenous infusions to increase plasma Zn(2+) and an infusion of the chelator, citrate, to decrease labile Zn(2+) in the plasma without altering total Zn(2+) content. In a model of mild surgical stress (unilateral femoral arterial ligation), plasma levels of total and labile Zn(2+) decreased significantly 24 h after the operation. Ultrafiltration of plasma into high- and low-molecular weight macromolecule fractionations indicated that binding capacity of zinc in the high-molecular weight fraction is impaired for the entire 24-h interval after induction of mild surgical stress. Affinity of the filtrate fraction was rapidly and reversibly responsive to anesthesia alone, decreasing significantly at 4 h and recovering at 24 h; in animals subjected to moderate surgical stress, this responsiveness was lost. These findings are the first reported measurements of labile Zn(2+) in the circulation in any form of mild systemic stress. Zinc undergoes substantial redistribution in the plasma as a response to surgical stress, leading to increased availability in lower molecular weight fractions and in its labile form.

Antioxidant pre-treatment prevents omeprazole-induced toxicity in an in vitro model of infectious gastritis.

Omeprazole is a mainstay of therapy for gastroesophageal reflux disease (GERD) and gastritis, and is increasingly used as an over-the-counter remedy for dyspepsia. Omeprazole acts by selectively oxidizing thiol targets in the gastric proton pump, but it also appears to be toxic to the gastric mucosa. We hypothesized that omeprazole toxicity is due to non-specific oxidation of cell structures other than the proton pump, and tested the efficacy of antioxidants to prevent omeprazole-induced toxicity in isolated rabbit gastric glands. Toxicity was measured by uptake and converstion of calcein-AM, following three hours of exposure to omeprazole and a non-selective thiol-oxidant, monochloramine. Intracellular concentration of Zn(2+) and the capacity to maintain luminal acidity were monitored using the fluorescent reporters fluozin-3 and Lysosensor DND-160, respectively. Both omeprazole and monochloramine caused marked reduction in cell viability. The toxicity of omeprazole was independent of monochloramine toxicity. The thiol reducing agent dithiothreitol protected gastric glands from injury. The oxidant scavenger Vitamin C also protected, and did not impair the anti-secretory effects of omeprazole. Thus, omeprazole toxicity appears to be oxidative and preventable with antioxidant therapy, including Vitamin C. Vitamin C may be a safe and efficacious addition to treatments requiring the use of PPIs.

Monochloramine-induced toxicity and dysregulation of intracellular Zn2+ in parietal cells of rabbit gastric glands.

Monochloramine (NH(2)Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate Ca(2+) homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([Zn(2+)](i)) in the gastric gland of the rabbit and that the increased Zn(2+) within the cell causes an independent decrease in cell viability. Changes in [Zn(2+)](i) were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [Zn(2+)](i) is increased in gastric glands exposed to NH(2)Cl and that elevated [Zn(2+)](i) decreases cell viability. Chelation of Zn(2+) with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of NH(2)Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [Zn(2+)](i) early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of NH(2)Cl.

Demand for Zn2+ in Acid-Secreting Gastric Mucosa and Its Requirement for Intracellular Ca2+

Background and Aims Recent work has suggested that Zn2+ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn2+ in gastric mucosa under baseline conditions and its regulation during secretory stimulation. Methods and Findings Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn2+ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, 70Zn2+, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of 70Zn2+ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn2+ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn2+ {[Zn2+]i} during exposure to standard extracellular concentrations of Zn2+ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn2+ increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca2+ {[Ca2+]i}. Uptake of Zn2+ was abolished following removal of extracellular Ca2+ or depletion of intracellular Ca2+ stores, suggesting that demand for extracellular Zn2+ increases and depends on influx of extracellular Ca2+. Conclusions This study is the first to characterize the content and distribution of Zn2+ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca2+ integrates basolateral demand for Zn2+ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.

Hemorrhagic Shock and Surgical Stress Alter Distribution of Labile Zinc within High and Low Molecular Weight Plasma Fractions

ABSTRACT Zinc ions (Zn2+) are essential for tissue repair following injury or stress. We hypothesize that during such stresses Zn2+ is redistributed to labile pools in plasma components. Here we tested this hypothesis using a novel assay to monitor labile Zn2+ in plasma in hemorrhagic shock. Adult rats in the shock group (S group) underwent hemorrhage and resuscitation. Blood samples were drawn at baseline and at 1, 4, and 24 h. The surgical control group (SC group) was anesthetized and instrumented, but not bled. Albumin, total Zn2+, and labile Zn2+ levels were assayed in plasma. Binding capacity for Zn2+ was assessed in high- and low-molecular-weight pools. Significant decreases in total Zn2+ were observed by 24 h, in both S and SC groups. Albumin levels were significantly reduced in the S group at 1 and 4 h but restored at 24 h; significant changes were not observed in other groups. In whole plasma, labile Zn2+ levels were stable initially in the S and SC groups, but declined at 24 h. In the high-molecular-weight pool, marked and significant impairment of binding was noted throughout all time periods following the shock period in the S group. Such changes were observed in the SC group of less intensity and duration. These experiments suggest that shock alters affinity of plasma proteins for Zn2+, promoting delivery to peripheral tissues during periods of increased Zn2+ utilization.