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MUCOSAL BIOLOGY

Intracellular Ca²⁺ and Zn²⁺ signals during monochloramine-induced oxidative stress in isolated rat colon crypts

¹Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and ²Division of Colon and Rectal Surgery, Department of Surgery, Mayo Clinic, Rochester, Minnesota

Submitted 8 November 2004 ; accepted in final form 25 May 2005

	ABSTRACT

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During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH₂Cl), to serve as agonists of Ca²⁺ and Zn²⁺ accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca²⁺ and Zn²⁺ in the cytoplasm. Responses to different concentrations of NH₂Cl (50, 100, and 200 µM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH₂Cl-induced increases in Ca²⁺ and Zn²⁺ in the cytoplasm. Exposure to NH₂Cl led to dose-dependent increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the range of 200–400 nM above baseline levels. Further studies indicated that NH₂Cl-induced accumulation of Ca²⁺ in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca²⁺ through store-operated channels. In addition, exposure to NH₂Cl resulted in dose-dependent and sustained increases in intracellular Zn²⁺ concentration ([Zn²⁺]_i) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca²⁺- and Zn²⁺-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca²⁺]_i and [Zn²⁺]_i. Under certain conditions, NH₂Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.

key words

In this study, we explored the ability of one class of oxidants, chloramines, to serve as agonists of Ca²⁺ and Zn²⁺ release to the cytoplasm of the colonocyte. The prototype in this class of oxidants, monochloramine (NH₂Cl), is produced through the reaction of neutrophil-derived hypochlorous acid (HOCl) with bacteria-derived ammonia (NH₃; see Refs. 25 and 63). NH₂Cl is relatively stable in aqueous environments and cell permeant (24, 25). Potential targets of NH₂Cl oxidation include molecules and structures in intracellular compartments as well as those on the cell membrane. Other chloramines species are generated by transfer of the oxidizing Cl· radical to amine groups of small organic acids such as taurine, lysine, or histamine (63). These chloramine species retain oxidant capacity but may not be able to permeate cell membranes. Molecular species capable of consuming or neutralizing the oxidant Cl· of chloramines include glutathione and other peptides and proteins (59) in which thiol (S-H) groups or clusters are integral to structure or enzymatic functions. Recent studies have implicated such thiol groups in structural proteins and enzymes that regulate intracellular homeostasis of divalent cations such as Ca²⁺ and Zn²⁺ (16, 46, 48, 77). These considerations led us to hypothesize that exposure to NH₂Cl may elicit a distinct profile of disturbances in intracellular divalent cation homeostasis in epithelial cells of the colon crypt.

	METHODS

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Solutions and reagents. Ringer solutions contained (in mM) 145 NaCl, 2.5 KH₂PO₄, 1.0 MgSO₄ or MgCl₂, 1 CaCl₂, 10 HEPES, and 10 glucose, pH = 7.4. Ca²⁺-free Ringer contained all of the compounds in standard Ringer except Ca²⁺, and, in addition, 500 µM EGTA was added. Intracellular buffer (ICB) contained (in mM) 125 KCl, 25 NaCl, 10 HEPES, 0.3 CaCl₂, 0.5 MgCl₂, 0.5 ATP, and 0.5 EGTA, pH = 7.25. Thapsigargin (THPS), ionomycin, and 1-hydroxypyridine-2-thione (pyrithione) were purchased from Sigma Chemical and used from stock solutions containing DMSO. Carbachol (CCh; Sigma), dithiothreitol (DTT; Sigma), thimerosal (Sigma), S-nitroso-N-acetyl-D,L-penicillamine, (SNAP; Sigma), N,N,N',N'-tetrakis(2-pyridyl-methyl) (TPEN; Molecular Probes), and 2-aminoethoxydiphenylborate (2-APB; Sigma) were dissolved in stock water and then brought up in Ringer (1:1,000) or dissolved directly in Ringer.

NH₂Cl was prepared as described previously (24, 62). Briefly, a 200-µl solution containing 500 mM NaOCl in water was added dropwise to 10 ml of 20 mM NH₄Cl and 5 mM Na₂HPO₄ in water at 0°C. This procedure resulted in a 5 mM NH₂Cl solution. Use of NH₂Cl was completed within 6 h of preparation, since we observed that it remained stable in Ringer solution at concentrations ranging from 50 to 200 µM with <10% loss of absorbance at 242 nm. TaurNHCl was generated under similar conditions by including taurine instead of NH₄Cl in the reaction mixture. Concentrations were verified by measuring absorbance in an ultraviolet spectrophotometer at 242, 292, and 252 nm for NH₂Cl, HOCl, and TaurNHCl, respectively. NH₂Cl concentration ([NH₂Cl]), HOCl concentration, and TaurNHCl concentration were then quantified using molar extinction coefficients reported previously (76). Solutions containing chloramines did not include and were not mixed with solutions containing DMSO to avoid direct consumption of oxidants that has been reported previously (65). We also performed in vitro studies indicating that DMSO can consume chloramines and, at high concentration (>1:100 vol/vol), interfere with measurements of Zn²⁺ in solution.

Crypt isolation. Anesthetic and procedures for killing used in these experiments were approved according to policies of Harvard Medical School. Male Sprague-Dawley rats (Charles River Laboratories) weighing 300 g were anesthetized and underwent laparotomy. The distal colon was identified, and a 1- to 2-cm segment was removed. The viscus was opened and then rinsed in cold Ringer solution. As described previously (13), the colon was incubated in a Ca²⁺ chelation solution (966 mM NaCl, 1.5 mM KCl, 10 mM HEPES, 10 mM Tris, 27 mM NaEDTA or NaEGTA, 45 mM sorbitol, 28 mM sucrose, and 0.1% BSA) for 30 min at room temperature. After chelation, the tissue was manually shaken to liberate the crypts. The tissue was removed, and the solution was centrifuged at 200 rpm for 2 min. The supernatant was removed, and the pellet was resuspended in Ringer solution.

Dye loading: imaging and ratiometric measurements. Fura 2-AM and magfura 2-AM (Molecular Probes, Eugene, OR) were diluted in DMSO to a stock concentration of 1 mM. Suspended in 1.5 ml Eppendorf tubes, crypts were loaded in the dark at room temperature in Ringer solution with 8–10 µM fura 2-AM for 30 min. Subsequently, crypts were rinsed two times at 5-min intervals with Ringer solution, mounted on cover slips coated with poly-D-lysine, and transferred to the microscope stage (Nikon TE-2000). For studies utilizing fluozin 3, crypts were loaded under similar conditions for 20–30 min with 4–8 µM fluozin 3-AM. Coverslips were placed in a perfusion chamber for imaging studies, superfused with Ringer solution at room temperature.

Studies were performed using monochromator-based excitation (Applied Scientific Instrumentation, Eugene, OR). Emitted light was collected at 520 ± 15 nm. During studies with fura 2, fluorescence was monitored by alternating excitation at 340 and 380 nm. Data are presented as a ratio of cation-sensitive intensity at 340 nm over cation-insensitive intensity at 380 nm. For studies utilizing fluozin 3, crypts were excited at 488 nm. Digital images of crypts were captured using a digital CCD camera (Hammamatsu ORCA-ER). Images were processed using compatible software (Universal Imaging, Downington, PA) to yield background-corrected pseudocolor images. Images were acquired every 10 s to minimize photobleaching. Contributions of autofluorescence were measured and were negligible.

Data collection, analysis, and statistics. Simultaneous fluorescence measurements from a whole crypt were obtained, since signals from individual cells could not be discerned at this level of magnification. It has been reported previously that the cells of the crypt function as a unit and thus that signals in individual cells are reflected in regional measurements. Regions of interest selected for analysis included the base of the crypt (the region of cells closest to the submucosa) and the apex (the region closest to the lumen). Although responses were more robust in basal regions, no clear differences emerged between regions in responses to ionophores or to NH₂Cl. Thus results are reported for signals collected from the entire crypt.

Concentrations of extracellular Ca²⁺ in calibration solutions (containing TPEN and EGTA) were calculated using the internet-based program maxchelator (http://www.stanford.edu/%7Ecpatton/webmaxcS.htm). Dissociation constants (K_d) for fura 2 and fluozin 3 were calculated using the equation of Grynkiewicz et al. (26) formulated for both intensity-based and ratiometric dyes (http://www.molecularprobes.com/html). Data were summarized and reported as means ± SE. Comparisons were performed using ANOVA for multiple or sequential comparisons, as indicated.

	RESULTS

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Monitoring [Ca²⁺]_i in isolated colon crypts and controlling for interfering heavy metal divalent cations. Previous reports have demonstrated the feasibility of using dyes in the fura 2 and fluo 3 classes to monitor [Ca²⁺]_i levels in colon crypts in response to physiological stimuli (8, 40, 41). These approaches depend on assumptions (54) that have not been reported for primary cell preparations such as the colon crypt. Shown in Fig. 1, A and B, are digital images of a colon crypt loaded with fura 2-AM, recorded in visible light and during fluorescence excitation at 380 nm. The first assumption is that the dye is localized almost completely within the cytpoplasm. Fluorescence intensity was monitored in crypts loaded with fura 2 at 340 and 380 nm, before and after exposure of crypts to 10 µM digitonin in ICB, an agent that permeabilizes the cell membrane without disturbing the integrity of organelles (31, 54). In five separate experiments, digitonin induced a marked decrease in fluorescence at both wavelengths, in all cases over 85%, indicating that fluorescence signals are the result of dye localized in the cytoplasm.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Digital images in grayscale of an isolated rat colon crypt, loaded with fura 2-AM (8 µM) for 25 min. A: low visible light background. B: fluorescence excitation at 380 nm. Original magnification: x30.

To evaluate such interference, we measured fura 2 signals during exposure of crypts to ionomycin, a cationophore able to translocate both Ca²⁺ and heavy metal divalent cations (15). In preliminary studies, crypts were exposed to 5 µM ionomycin in the presence of no added Ca²⁺ and 0.5 mM EGTA ([Ca²⁺] 1 nM). Exposure to ionomycin rapidly increased the excitation ratio, indicating release of divalent cations to the cytoplasm. In six experiments, the interval and magnitude of recovery to baseline was highly variable. Such a response is characteristic of binding of the dye to a ligand of higher affinity, for example, Zn²⁺. To evaluate this possibility, we monitored responses to ionomycin and varying [Ca²⁺] in the presence of TPEN, a known chelator for Zn²⁺ (K_d 10^–15 M) and other metal divalent cations (1). TPEN also exhibits a very low affinity for Ca²⁺ (K_d 100 µM; see Refs. 1 and 21), thereby screening out contributions of interfering metal cations while permitting fura 2 to respond to cytoplasm Ca²⁺ signals in the physiological range (100 nM-1 µM; see Ref. 29). As shown in Fig. 2A, the presence of 20 µM TPEN permitted direct correlation of fura 2 signals to [Ca²⁺]_i. We found the greatest responsiveness in the range of 100–400 µM, with diminished responsiveness both above and below this range. As shown in Fig. 2B, the responses within this range provide a method for in situ calibration that controls for contributions from interfering metal polyvalent cations. Inserting the data in the relationship of Grynkiewicz et al. (26), we calculate that, in situ in the colonic crypt, the K_d of fura 2 for Ca²⁺ is 282 ± 32 nM (average ± SE, n = 9). This value is somewhat higher than those reported previously in cultured cells but quite close to those reported for primary epithelial cell preparations such as the gastric gland (26, 27, 54). Using similar methods in studies of five isolated colon crypts, we determined that the in situ K_d of fura 2 for Zn²⁺ is 4.7 ± 0.6 nM (average ± SE), quite close to reported values (27). In these studies (n = 5 crypts), fura 2-loaded crypts were exposed to 40 µM pyrithione, a heavy metal specific ionophore, in 0 Ca²⁺ Ringer with varying concentrations of Zn²⁺ (0, 2.5, 5, 7.5, 10, and 1,000 nM). Experiments were also undertaken to ensure that pyrithione does not transport Ca²⁺ in the cytoplasm from either extracellular solution or from the cellular organelles (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Fura 2 signals in EGTA-isolated colon crypts during incremental increases in Ca2+ concentration ([Ca2+]). Crypts were exposed to [Ca2+] varying from 100 to 400 nM in the presence of ionomycin (10 µM) and N,N,N',N'-tetrakis(2-pyridyl-methyl) (TPEN, 20 µM). A: recording of fura 2 fluorescence in an individual crypt. , Individual data points collected every 10 s. B: summary of calibration curves in 9 crypts using the same protocol as in A. Results are expressed as mean ± SE fluorescence excitation ratios (340/380 nm). With the use of averaged values in B, Kd of fura 2 for Ca2+ is 282 ± 32 nM.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of TPEN on "pure" intracellular Ca2+ signals monitored by fura 2. Crypts were loaded with fura 2-AM and then exposed to Ringer solution containing 0 Ca2+ and 0.5 mM EGTA in the absence () or presence () of 20 µM TPEN. The presence of TPEN had no significant effect on signals generated during exposure to 100 µM carbachol (CCh) and 1 µM thapsigargin (THPS), which irreversibly empties intracellular stores. Also, TPEN had no significant effect on the magnitude of capacitative entry, monitored when extracellular Ca2+ was restored. Each line represents a mean of 5 individual experiments and expresses the mean ± SE of individual data points (10-s intervals) of the excitation ratio (340/380 nm).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Measurements of fura 2 signals during exposure of colon crypts to NH2Cl. A: recording in an individual crypt exposed to 200 µM NH2Cl. Recording begins with crypts perfused by standard Ringer solution, then exposure to NH2Cl (5 min), and then standard Ringer. Note absence of reversibility. B: summary of responses to monochloramine at different doses (50, 100, and 200 µM). Data represent ratio before (open bars), during (black bars), and after (shaded bars) exposure to NH2Cl. Results are expressed as means ± SE, with the y-axis indicating the excitation ratio (340/380 nm). *P < 0.01 compared with baseline levels by ANOVA.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Measurements of fura 2 signals during exposure of colon crypts to NH2Cl, in the absence of extracellular Ca2+. A: averaged responses to NH2Cl at different doses [0 (control), 50, 100, and 200 µM). Each line represents responses in 5 or 6 separate experiments, each conducted in exactly the same sequence. Each experiment started with crypts perfused by standard Ringer, then exposure to 0 Ca2+-Ringer (0.5 mM EGTA), exposure to NH2Cl, removal of NH2Cl, and then restoration of Ca2+-Ringer. Each data point represents the mean ± SE of responses at each time point. B: key time points just before the peak response (open bars), during the peak response (black bars), and stablization after removal of NH2Cl (shaded bars). Results are expressed as means ± SE, with the axis on left indicating the excitation ratio (340/380 nm). *P < 0.01 compared with baseline levels by ANOVA. Arrows indicate where solutions change.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effects of TPEN on monochloramine-induced changes in fura 2 signals. , Colon crypts exposed to 0 Ca2+ Ringer followed by addition of 200 µM NH2Cl; , colon crypts exposed to 0 Ca2+ Ringer containing 20 µM TPEN, followed by addition of 200 µM NH2Cl. Each data point represents the mean ± SE of 5 experiments. Slopes of the increase (after addition of NH2Cl) are significantly different (P < 0.01 by linear regression). Note the exaggerated response to restoration of standard Ringer with physiological [Ca2+] and lack of inhibition of extracellular calcium entry by TPEN.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effects of TPEN on monochloramine-induced changes in fura 2 signals. , Colon crypts exposed to 0 Ca2+ Ringer followed by addition of 200 µM NH2Cl; , colon crypts exposed to 0 Ca2+ Ringer followed by addition of 200 µM NH2Cl. At the peak of the NH2Cl effect, crypts were then exposed to 20 µM TPEN. Slopes of the descent in the signal after NH2Cl are significantly different (P < 0.05 by linear regression).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Fura 2 signals were monitored during exposure to 200 µM NH2Cl under baseline conditions (left), after 12 min irreversible depletion of intracellular stores by treatment with CCh (100 µM) and THPS (1 µM) (middle), and after depletion of stores and 5 min exposure to TPEN (20 µM) (right). *P < 0.01 compared with pre-NH2Cl baseline. P < 0.05 vs. the response [change in () signal] to that observed under control conditions.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Effects of 2-aminoethoxydiphenylborate (2-APB) on store-operated entry induced by NH2Cl. Top: superimposed recordings from fura 2-loaded crypts from the same harvest. Both crypts were exposed to 200 µM NH2Cl, in a protocol similar to that used in Fig. 6, with similar responses. As before, 20 µM TPEN was present to screen out contributions from heavy metal cations. Bottom: after removal of NH2Cl, glands were exposed to 2-APB or vehicle (ethanol 1:1,000), and then extracellular Ca2+ was restored. In the presence of 2-APB, increases in intracellular [Ca2+] ([Ca2+]i; ratio) were diminished significantly; n = 7 crypts in each group. *P < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 10. A: digital images in grayscale of an isolated rat colon crypt loaded with fluozin 3-AM (4 µM) for 25 min. Fluorescence excitation was at 488 nm. B: recording of fluozin 3 fluorescence during exposure to different concentrations of Zn2+ buffered with EGTA in 0 Ca2+ Ringer with 40 µM sodium pyrithione. Fluorescence is expressed in arbitrary units of intensity normalized to starting values (F/Fo). C: summary of calibrations in 6 individual crypts. With the use of averaged values in C, Kd of fluozin 3 for Zn2+ is 4 ± 0.3 nM.

Effects of NH₂Cl on [Zn²⁺]_i in isolated colon crypts. To evaluate the effects of NH₂Cl, we exposed isolated colon crypts to solutions containing [NH₂Cl] of 50, 100, and 200 µM. As shown in Fig. 11A, rapid increases were observed in response to NH₂Cl, indicating that exposure to NH₂Cl causes a marked release of intracellular pools of Zn²⁺ within the colonocyte. The addition of pyrithione in a Zn²⁺-free solution and in a 1-µM Zn²⁺ solution shows the ability of the ionophore to remove Zn²⁺ and the maximum signal capable under these experimental procedures, respectively. These signals were similarly quenched when TPEN was added to the perfusate (n = 4 experiments, data not shown), providing additional assurance that the signals were attributable to increases in [Zn²⁺]_i. As shown in Fig. 11B, these effects were dose dependent. Based on calibration responses shown in Fig. 10C, it appears that labile [Zn²⁺]_i may increase to as much as 4–6 nM during exposure to 200 µM NH₂Cl.

View larger version (24K): [in this window] [in a new window]   Fig. 11. Measurements of fluozin 3 signals during exposure of colon crypts to NH2Cl. A: recording in an individual crypt exposed to 200 µM NH2Cl in 0 Ca2+ Ringer. Recording begins with crypts perfused by standard Ringer solution, then removal of Ca2+, followed by exposure to NH2Cl (5 min), and recovery in 0 Ca2+ Ringer. Sodium pyrithione (Na-pyr, 40 µM) was added in the absence of Zn2+ followed by the addition of 1 µM Zn2+. B: summary of responses to monochloramine at different doses (50, 100, and 200 µM). Results are expressed as means ± SE, with the y-axis indicating fluorescence intensity normalized to starting values (F/F0). Data represent recordings before (open bars), during (black bars), and after (shaded bars) removal of NH2Cl. *P < 0.05 compared with treatment before NH2Cl.

Additional control studies were performed to evaluate the possibility that NH₂Cl or its reactants might directly alter fluorescence properties of fura 2 or fluozin 3. In a cell-free chamber, each dye was placed in its free acid form, in the presence of EGTA-buffered solutions containing either 500 nM Ca²⁺ and 20 µM fura 2 or 2 nM Zn²⁺ and 20 µM fluozin 3. Addition of NH₂Cl, HOCl, or NH₃ all failed to alter baseline levels of fluorescence at the individual exciting wavelengths (340 and 380 nm for fura 2; 488 nm for fluozin 3). We also found no response to 100 µM peroxide, a precursor used by the neutrophil to generate HOCl. In addition, these reagents did not interfere with changes in fluorescence caused by doubling of divalent cation concentrations (data not shown).

We also performed studies to determine whether such effects might be observed in response to other recognized thiol oxidants, such as thimerosal (5, 45) and nitric oxide donors SNAP or 3-morpholinosydnonimine N-ethylcarbamide (SIN-1; see Refs. 18 and 19). In fura 2-loaded crypts (n = 4) exposed to thimerosal (100 µM), no responses were observed. In crypts exposed to the NO donor SNAP (1 mM), small but significant increases in fura 2 fluorescence were observed (n = 5 crypts, fura 2 excitation ratio 340/380 increasing from 1.00 ± 0.01 to 1.25 ± 0.05, P < 0.005; Fig. 12). Lower doses of SNAP (100 µM, n = 5) had no appreciable effect. These increases were not abolished in the presence of TPEN (n = 3 crypts). In addition, when fluozin 3-loaded crypts were exposed to SNAP (1 mM), little or no increase in fluorescence was observed. These findings indicate that SNAP-induced increases in the fura 2 ratio are not attributable to accumulation of polyvalent cations such as Zn²⁺ and that Ca²⁺ is the accumulating ion. In other studies, the NO donor SIN-1 also elicited small but highly variable responses in fura 2-loaded crypts (n = 4). These observations indicate that release and accumulation of Ca²⁺ and Zn²⁺ are not observed uniformly in response to all thiol oxidants.

View larger version (12K): [in this window] [in a new window]   Fig. 12. Fura 2 fluorescence recording from an individual fura 2-loaded crypt exposed to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP).

View larger version (21K): [in this window] [in a new window]   Fig. 13. Thiol reduction as a means of preventing or reversing NH2Cl effects. A: recording in an individual crypt exposed to 200 µM NH2Cl in 0 Ca2+ Ringer already containing 1 mM dithiothreitol (DTT). DTT was then removed with NH2Cl remaining present. The crypt was allowed to recover in 0 Ca2+ Ringer, followed by addition of standard Ringer. B: recording in an individual crypt exposed to 1 mM DTT after initiation of responses to 200 µM NH2Cl in 0 Ca2+ Ringer. NH2Cl was later removed with DTT remaining present. Again, the crypt was allowed to recover in 0 Ca2+ Ringer, followed by addition of standard Ringer.

	DISCUSSION

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To our knowledge, measurements of [Zn²⁺]_i have not been reported for epithelial cells of the colon mucosa. Moreover, previous reports utilizing fluorescence-based measurements of [Ca²⁺]_i in colonocytes have not taken into account contributions of other divalent cations, such as Zn²⁺. To monitor isolated changes in [Ca²⁺]_i in the isolated colon crypt, we used a well-recognized, ratiometric fluorescent reporter, fura 2. In the presence of TPEN, which chelates non-Ca²⁺ metal divalent cations without interfering with levels of [Ca²⁺]_i, fura 2 would monitor "pure" Ca²⁺ signaling responses to neurohumoral or pathological stimuli. To monitor changes in [Zn²⁺]_i directly, we used fluozin 3, a dye recently reported to be useful for measurements in the subnanomolar to nanomolar range.

Our studies indicate that NH₂Cl causes dose-dependent increases in [Ca²⁺]_i in native colon crypts of the rat. Further studies indicate that NH₂Cl-induced accumulation of Ca²⁺ in the cytoplasm is the result of release from intracellular stores, as well as store-operated entry from extracellular fluid. The duration of these increases is sustained beyond those associated with physiological stimuli (8, 41), but are partly reversible, even during exposure to NH₂Cl concentrations of up to 200 µM. We also noted that it was technically feasible to characterize these responses only when contributions of other divalent cations were screened out using the heavy metal chelator TPEN. In the presence of 20 µM TPEN, the increases in [Ca²⁺]_i are in the range of concentrations that are reported as physiological signals (11, 17, 41, 54). A comparison between responses to physiological agonists (Fig. 3) and to NH₂Cl (Fig. 6) indicates that, when contributions of other divalent cations are taken into account, the responses (perhaps 200–300 nM above baseline) lie within the range expected in response to neurohumoral stimuli.

We also found that exposure of crypts to NH₂Cl leads to dose-dependent increases in [Zn²⁺]_i in the range of 3–5 nM, which are sustained and not easily reversed. These effects on [Ca²⁺]_i and [Zn²⁺]_i are prevented by pretreatment with DTT, which shields intracellular thiol groups from oxidation by chlorinated oxidants. In addition, these effects are partially reversed by exposure to DTT, effects not attributable to chelation of Ca²⁺ or Zn²⁺. These findings indicate that thiol groups in Ca²⁺- and Zn²⁺-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca²⁺]_i and [Zn²⁺]_i. These findings offer the possibility that there is a range of concentrations over which NH₂Cl may not be toxic but instead stimulates release of physiological signals of oxidant stress in the colonocyte.

These studies raise three issues for discussion. The first issue is a technical one, namely, the strengths and limitations of fluorescence methods used to monitor intracellular divalent cation signals. Ca²⁺-sensing fluorescent indicator dyes all are responsive to other polyvalent cations. Some potentially interfering ions (such as Cd²⁺, Ba²⁺, and La³⁺) are excitatory (i.e., elicit responses similar to Ca²⁺) but are not expected to interfere because they can only be added exogenously. In addition, some toxic cations (Mn²⁺, Cd²⁺, and Pb²⁺) cause quenching or shifts in excitation spectra, rather than amplification of intensities; thus, they do not elicit responses similar to those of Ca²⁺. Potentially interfering divalent cations include Fe²⁺, Fe³⁺, Cu²⁺, and Zn²⁺, which might be released from intracellular pools. The most widely used fluorescent reporter, fura 2, is quenched in response to Cu²⁺, Fe²⁺, and Fe³⁺, but it responds to Zn²⁺ in a similar manner to Ca²⁺ (38). In fact, previous reports and manufacturer specifications indicate that, in vitro, fura 2 is more responsive to Zn²⁺ (K_d 3–15 nM) than it is to Ca²⁺ (K_d 150–300 nM; see Refs. 27, 38, and 54). Our calibration studies confirm these ranges of sensitivities to Ca²⁺ and Zn²⁺ in situ in cells of the rat colon crypt. These considerations suggest that, when fura 2 is used to monitor changes in [Ca²⁺], signals may include intracellular accumulation of Zn²⁺ in the nanomolar range.

Our studies demonstrate that fura 2 monitors increases in [Zn²⁺]_i under two sets of conditions. First, it appears that Zn²⁺ accumulates in the cytoplasm during exposure of the crypt to ionomycin, an ionophore frequently used to release Ca²⁺ from intracellular stores or to equilibrate Ca²⁺ in the cytoplasm with that in the extracellular fluid. Second, we observed release of Zn²⁺ during exposure to NH₂Cl, an oxidant that preferentially targets proteins containing thiolate clusters that bind or sequester heavy metal divalent cations. In both conditions, there are three considerations on which we base the conclusion that Zn²⁺ is the interfering ion. First, of the likely contaminating heavy metals, only Zn²⁺ elicits fluorescence responses similar to those of Ca²⁺. Second, during exposure of crypts to ionomycin or NH₂Cl, fura 2 signals are reduced in the presence of TPEN, a chelator with profound affinity for Zn²⁺ (K_d 10^–15 M) and other heavy metals but not Ca²⁺ or Mg²⁺. Third, exposure to ionomycin and NH₂Cl led to increases in fluorescence in crypts loaded with fluozin 3, a reporter with a high degree of preference for Zn²⁺ at nanomolar concentrations and virtual insensitivity to Ca²⁺ in the submicromolar range. Our studies do not necessarily exclude release of other divalent cations (for example Fe²⁺, Fe³⁺, or Cu²⁺) during exposure to ionomycin or NH₂Cl. However, they would argue that the release of Zn²⁺, which excites fura 2 fluorescence with high affinity, outweighs the effects of other metal polyvalent cations that would tend to quench fura 2 signals. Our findings also send a fundamental message that, in using fluorescent reporters to explore changes in [Ca²⁺]_i during exposure to oxidants, toxins, and relatively uncharacterized neurohumoral agonists, it is important to control or take into account accumulation of interfering polyvalent cations.

The second issue raised by these studies involves the mechanisms by which NH₂Cl causes accumulation of Ca²⁺ and Zn²⁺ within the cytoplasm of the colon crypt cell. With respect to [Ca²⁺]_i, our studies indicate that NH₂Cl-induced accumulations are the result of release of Ca²⁺ from intracellular stores and influx from the extracellular microenvironment. When crypts were perfused with 0 Ca²⁺ Ringer and pretreated with CCh/THPS, exposure to NH₂Cl diminished the increases in [Ca²⁺]_i, implying that THPS-sensitive stores are the major intracellular sources for the Ca²⁺ signal. These responses are attributable to thiol modification of Ca²⁺ transport processes, since DTT prevented and reversed effects of NH₂Cl on fura 2 signals.

The molecular basis for oxidant-induced release of Ca²⁺ from intracellular stores is not clear. In addition to direct modification of Ca²⁺ release channels in the membrane of the endoplasmic reticulum (16, 77), thiol oxidants might alter receptors regulating release of Ca²⁺ from stores to the cytoplasm. In this regard, both InsP₃ receptors (44) and ryanodine receptors (37) have been identified in colon crypts, the former having also been shown to support Ca²⁺-dependent anion secretion (37). In other tissues, functional modification of either receptor has been demonstrated after exposure to thiol oxidants (14, 45), the latter in response to chloramines (14). These considerations suggest that NH₂Cl may find a broad spectrum of targets in the apparatus that regulates store emptying. Future studies, beyond the scope of the current report, may thus provide insight into the role that thiol redox state plays in regulating both store emptying and refilling.

Subsequent responses to solutions containing physiological levels of Ca²⁺, however, make it clear that entry of extracellular Ca²⁺ is responsible for a major component of the NH₂Cl-induced signal (Figs. 6 and 7). Entry of extracellular Ca²⁺ might be because of influx through capacitative entry, activated when CCh- and THPS-sensitive stores are released (29–31). Alternatively, NH₂Cl-induced entry of extracellular Ca²⁺ might be independent of store emptying. In the present study, we found that 2-APB inhibits a significant component of NH₂Cl-stimulated entry of extracellular Ca²⁺, arguing that NH₂Cl elicits its effects on extracellular influx through its effects on store depletion. It should be acknowledged, however, that, in the crypt configuration, it is difficult to directly evaluate the linkage between NH₂Cl-induced store emptying and influx of extracellular Ca²⁺. It is possible that exposure to NH₂Cl may disengage store emptying and the "capacitative" entry that normally leads to store refilling. In cultured cell models, this separation of function (store release and store refilling) has been observed as a result of thiol modification through S-nitrosylation or alkylation (16, 42). Alternatively, exposure to NH₂Cl might modify Ca²⁺ entry mechanisms that are independent of store emptying; in such cases, thiol modification has not been implicated (77). Further studies using reporters of intracellular stores may provide insight into these different possibilities (29–31). At the very least, our studies indicate that influx of extracellular Ca²⁺ supplies a major component of the sustained increase in [Ca²⁺]_i caused by NH₂Cl. It seems likely that the activation of such influx is at least partially independent of the effects of NH₂Cl on emptying of intracellular Ca²⁺ stores and is the result of thiol modification.

With respect to increases in [Zn²⁺]_i, release of Zn²⁺ might be observed from specific Zn²⁺-enriched subcellular compartments or from cytoplasm pools of Zn²⁺-binding proteins that are susceptible to thiol modification. Subcellular compartments enriched in labile Zn²⁺ include acidic secretory compartments (20, 21) and under some conditions mitochondria (69). Cytoplasm pools of Zn²⁺-binding proteins include metallothioneins, glutathione, heat shock proteins, and other peptides enriched in cysteine or methionine (32, 46, 48, 49). A number of studies have indicated that Zn²⁺ may be released by accumulation of oxidants that tend to attack thiol clusters in metallothioneins (32, 49). Currently, very little is reported on the mechanisms by which Zn²⁺ is transported in the colonocyte and distributed to intracellular pools and subcellular compartments. The current studies establish a reliable method for monitoring changes in intracellular [Zn²⁺] in colon crypts under physiological conditions and during oxidative stress. In addition, they provide evidence that NH₂Cl, and other oxidants that preferentially target S-H groups, may be highly effective in releasing Zn²⁺ to the cytoplasm of cells in the colon crypt.

The final issue raised by our studies involves the consequences of oxidant-induced increases in [Ca²⁺]_i and [Zn²⁺]_i in the colon crypt. We find that exposure to NH₂Cl elicits significant, but not necessarily unphysiological, increases in both [Ca²⁺]_i and [Zn²⁺]_i.Our studies also suggest that chloramines may be considerably more powerful in eliciting these responses than are other recognized classes of thiol oxidants, most notably donors of nitric oxide. These observations raise intriguing questions regarding the structural specificity of different reactive nitrogen species, their targets, and potential intracellular pools buffering against their activity (thiol-rich molecules such as glutathione, metallothionein, etc). It is well recognized that increases in [Ca²⁺]_i trigger secretion in colon crypts (8, 51, 72, 73) and inhibit absorption in surface epithelium (28, 79), considered a "flushing" response that clears luminal bacteria and other noxious agents away from the mucosal surface (53, 57). In diverse in vitro cell culture models, agonist-stimulated increases in [Ca²⁺]_i enhance mitochondrial respiration and substrate utilization (34, 66). In excess, increases in [Ca²⁺]_i induce mitochondrial depolarization and the initiating events of apoptosis and/or cell necrosis (12, 56). In addition, studies in tissues outside the gastrointestinal tract have suggested that key metabolic and protective pathways are also sensitive to increases in [Zn²⁺]_i. These pathways include enzymes of anaerobic glycolysis (4, 70) and the aerobic tricarboxylic acid cycle (10), specific isoforms of protein kinase C (6, 9, 71), downstream effects of cAMP signaling (36), modulation of mitochondrial respiration (69), and preexecution steps of apoptosis (7, 33). In the aggregate, these studies have suggested that uncontrolled accumulation of Ca²⁺ and Zn²⁺ may exacerbate mucosal injury. However, they also have offered the possibility that intracellular release of these divalent cations, coordinated and in moderation, may elicit responses that prevent, arrest, or possibly reverse inflammation-induced injury. The current set of observations provides a frame of reference for understanding the potential downstream effects of sustained increases in intracellular levels of [Ca²⁺]_i and [Zn²⁺]_i, by providing quantitative estimates of the magnitude and time course of these increases.

In summary, we have adapted fluorometric approaches for monitoring changes in [Ca²⁺]_i and [Zn²⁺]_i in isolated crypts of the rat colon during exposure to oxidant stress. Our studies indicate that experimental approaches for monitoring [Ca²⁺]_i should control or take into account contributions from interfering polyvalent cations. When these factors are taken into account, it appears that NH₂Cl elicits increases in [Ca²⁺]_i that are sustained but certainly not much higher than those expected from normal signaling processes. These increases reflect contributions from emptying of physiologically regulated intracellular stores, as well as entry of extracellular Ca²⁺, most likely through store-operated channels. In addition, increases in [Zn²⁺]_i are observed in response to NH₂Cl and probably reflect release from cytoplasm pools of metal-binding proteins such as metallothionein. Increases in [Zn²⁺]_i may thus act as signals of oxidative stress, potentially activating downstream responses that are protective and anti-apoptotic. Further studies will determine the role played by these signaling responses to unique thiol-directed oxidants such as NH₂Cl in injury and protection in acute colitis.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-44571 (to D. I. Soybel), by the Brigham Surgical Group Foundation (to D. I. Soybel), and by a Howard Hughes Medical Institute Student Fellowship (to A. M. Wieland).

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. I. Soybel, Dept. of Surgery, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (e-mail: dsoybel{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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