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Intracellular Ca 2+ and Zn 2+ signals during monochloramine-induced oxidative stress in isolated rat colon crypts

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作者:Robert R. Cima,, J. Matthew Dubach, Aaron M. Wieland, Breda M. Walsh, and David I. Soybel    作者单位:1 Department of Surgery, Brigham and Women‘s Hospital and Harvard Medical School, Boston, Massachusetts; and 2 Division of Colon and Rectal Surgery, Department of Surgery, Mayo Clinic, Rochester, Minnesota

【摘要】  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 (NH 2 Cl), to serve as agonists of Ca 2+ and Zn 2+ 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 Ca 2+ and Zn 2+ in the cytoplasm. Responses to different concentrations of NH 2 Cl (50, 100, and 200 µM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH 2 Cl-induced increases in Ca 2+ and Zn 2+ in the cytoplasm. Exposure to NH 2 Cl led to dose-dependent increases in intracellular Ca 2+ concentration ( i. Under certain conditions, NH 2 Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.

【关键词】  key words


INFLAMMATORY BOWEL DISEASES are thought to result from inappropriate activation of the mucosal immune system in response to normal flora ( 58, 60, 68 ). During acute exacerbations of these illnesses, reactive species are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In inflamed human tissues and experimental models of colitis, oxidants and reductants derived from oxygen and nitrogen play a critical role in toxicity to microbes and injury to the "bystander" colon mucosa ( 2, 3, 39, 50, 58, 80 ). Presumed mechanisms of oxidant-induced injury of the epithelium include direct chemical attack on cell membranes and intracellular compartments, with consequences that include depolymerization of mucin; peroxidation of lipids; oxidation of structural proteins, enzymes, and carbohydrates; disorganization of DNA; and increased mucosal permeability. In addition, exposure to oxidants may activate intracellular signaling pathways that may exacerbate injury or, conversely, initiate protective responses in the inflamed mucosa. Affected pathways include protein kinase A and C activities, expression of heat-shock proteins, and modulation of NF- B and effector pathways in apoptosis ( 24, 35, 52, 55, 58, 74 ). Of special interest is oxidant-induced release of the divalent cations, Ca 2+ and Zn 2+, to the cytoplasm. When uncontrolled and in excess, such divalent cation signals can exacerbate tissue injury ( 10, 56, 67 ). When coordinated and in moderation, however, such signals may elicit secretory or protective responses that would prevent or arrest inflammation-induced injury ( 36, 69, 72 ).


In this study, we explored the ability of one class of oxidants, chloramines, to serve as agonists of Ca 2+ and Zn 2+ release to the cytoplasm of the colonocyte. The prototype in this class of oxidants, monochloramine (NH 2 Cl), is produced through the reaction of neutrophil-derived hypochlorous acid (HOCl) with bacteria-derived ammonia (NH 3; see Refs. 25 and 63 ). NH 2 Cl is relatively stable in aqueous environments and cell permeant ( 24, 25 ). Potential targets of NH 2 Cl 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 Ca 2+ and Zn 2+ ( 16, 46, 48, 77 ). These considerations led us to hypothesize that exposure to NH 2 Cl may elicit a distinct profile of disturbances in intracellular divalent cation homeostasis in epithelial cells of the colon crypt.


METHODS


Solutions and reagents. Ringer solutions contained (in mM) 145 NaCl, 2.5 KH 2 PO 4, 1.0 MgSO 4 or MgCl 2, 1 CaCl 2, 10 HEPES, and 10 glucose, pH = 7.4. Ca 2+ -free Ringer contained all of the compounds in standard Ringer except Ca 2+, and, in addition, 500 µM EGTA was added. Intracellular buffer (ICB) contained (in mM) 125 KCl, 25 NaCl, 10 HEPES, 0.3 CaCl 2, 0.5 MgCl 2, 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.


NH 2 Cl 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 NH 4 Cl and 5 mM Na 2 HPO 4 in water at 0°C. This procedure resulted in a 5 mM NH 2 Cl solution. Use of NH 2 Cl 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 NH 4 Cl in the reaction mixture. Concentrations were verified by measuring absorbance in an ultraviolet spectrophotometer at 242, 292, and 252 nm for NH 2 Cl, HOCl, and TaurNHCl, respectively. NH 2 Cl concentration (), 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 1:100 vol/vol), interfere with measurements of Zn 2+ 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 Ca 2+ 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 NH 2 Cl. Thus results are reported for signals collected from the entire crypt.


Concentrations of extracellular Ca 2+ 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


Monitoring 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.


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: x 30.


The second assumption of such fluorescence-based methods is that contributions of other interfering cations are negligible or can be controlled. All useful reporters respond to multiple divalent cations, although with different profiles of response ( 1, 26 ). In this regard, published reports suggest that, in cell-free systems, fura 2 increases fluorescence in response to Ca 2+ ( K d 145 nM), Zn 2+ ( K d 3 nM), and other metals not likely to interfere with fluorescence measurements, such as Cd 2+ ( K d 1 pM) and Co 2+ ( K d 9 nM; see Ref. 27 ). Responses to physiologically significant metals such as Cu 2+ and Fe 2+ are recognized but either cause quenching of signals or elicit responses opposite to those of Ca 2+ and Zn 2+ ( 1, 27, 38 ). However, release of such cations from intracellular pools or subcellular compartments could interfere with measurements of Ca 2+ concentration () when crypts are exposed to potentially toxic agents.


To evaluate such interference, we measured fura 2 signals during exposure of crypts to ionomycin, a cationophore able to translocate both Ca 2+ and heavy metal divalent cations ( 15 ). In preliminary studies, crypts were exposed to 5 µM ionomycin in the presence of no added Ca 2+ and 0.5 mM EGTA ( 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. 2 B, 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 Ca 2+ 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 Zn 2+ 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 Ca 2+ Ringer with varying concentrations of Zn 2+ (0, 2.5, 5, 7.5, 10, and 1,000 nM). Experiments were also undertaken to ensure that pyrithione does not transport Ca 2+ in the cytoplasm from either extracellular solution or from the cellular organelles (data not shown).


Fig. 2. Fura 2 signals in EGTA-isolated colon crypts during incremental increases in Ca 2+ concentration ( 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, K d of fura 2 for Ca 2+ is 282 ± 32 nM.


We next performed studies to evaluate whether the presence of TPEN might interfere with the ability of fura 2 to monitor physiological increases in signals elicited by CCh/THPS or during capacitative entry. Together, these studies indicate that TPEN may be used to screen out contributions of other heavy metal cations while preserving the intracellular Ca 2+ signals that are monitored by fura 2.


Fig. 3. Effect of TPEN on "pure" intracellular Ca 2+ signals monitored by fura 2. Crypts were loaded with fura 2-AM and then exposed to Ringer solution containing 0 Ca 2+ 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 Ca 2+ 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).


Effects of NH 2 Cl on at 50, 100, and 200 µM. As summarized in Fig. 4, A and B, dose-dependent and largely irreversible increases were observed in the fluorescence excitation ratio. These basic observations led us to perform studies to determine the sources of these signals, including the contributions of 1 ) extracellular Ca 2+, 2 ) intracellular pools of non-Ca 2+ heavy metal divalent cations, and 3 ) physiologically regulated intracellular Ca 2+ stores.


Fig. 4. Measurements of fura 2 signals during exposure of colon crypts to NH 2 Cl. A : recording in an individual crypt exposed to 200 µM NH 2 Cl. Recording begins with crypts perfused by standard Ringer solution, then exposure to NH 2 Cl (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 NH 2 Cl. 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.


In the first set of studies, Ca 2+ was removed from the perfusates, to which 0.5 mM EGTA was added to lower i under baseline conditions ( 17 ). During exposure to NH 2 Cl, the signal increased dose dependently to a plateau but then partially reversed when NH 2 Cl was withdrawn. The peak effects were greatly diminished compared with those observed in the presence of extracellular Ca 2+ ( Fig. 4 ), indicating that a significant component of the response to NH 2 Cl is because of influx of extracellular Ca 2+.


Fig. 5. Measurements of fura 2 signals during exposure of colon crypts to NH 2 Cl, in the absence of extracellular Ca 2+. A : averaged responses to NH 2 Cl 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 Ca 2+ -Ringer (0.5 mM EGTA), exposure to NH 2 Cl, removal of NH 2 Cl, and then restoration of Ca 2+ -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 NH 2 Cl (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.


In the second set of studies, we monitored fura 2 signals in crypts exposed to NH 2 Cl in the presence of TPEN. Crypts were first exposed to 0 Ca 2+ Ringer and 20 µM TPEN to eliminate contributions from extracellular Ca 2+ and to chelate labile metal cations present in intracellular pools, respectively. Crypts were then exposed to 200 µM NH 2 Cl for 5 min, allowed to recover in 0 Ca 2+ Ringer, and then exposed to standard Ca 2+ -Ringer. As shown in Fig. 6, preexposure of crypts to TPEN significantly reduced and delayed the peak effect of NH 2 Cl, indicating that a significant component of the NH 2 Cl-induced signal is the result of heavy metal divalent cations. To further characterize the sensitivity to TPEN, crypts were exposed to NH 2 Cl and then TPEN at the peak of the response. As shown in Fig. 7, exposure to TPEN at the peak response to NH 2 Cl caused a significantly more rapid dissipation in the signal. These findings indicate that TPEN chelates labile metal divalent cations released by NH 2 Cl and confirm that the fura 2 response is the result of release of both Ca 2+ and non-Ca 2+ metal divalent cations from within the cell.


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


Fig. 7. Effects of TPEN on monochloramine-induced changes in fura 2 signals., Colon crypts exposed to 0 Ca 2+ Ringer followed by addition of 200 µM NH 2 Cl;, colon crypts exposed to 0 Ca 2+ Ringer followed by addition of 200 µM NH 2 Cl. At the peak of the NH 2 Cl effect, crypts were then exposed to 20 µM TPEN. Slopes of the descent in the signal after NH 2 Cl are significantly different ( P < 0.05 by linear regression).


We then performed studies to determine whether intracellular pools of Ca 2+ released by NH 2 Cl might include physiologically regulated intracellular stores. To deplete these stores, crypts were perfused in 0 Ca 2+ Ringer solution alone or containing, in addition, 100 µM CCh and 1 µM THPS. Crypts were then exposed to 200 µM NH 2 Cl. As shown in Fig. 8, there was a significant decrease in the response to NH 2 Cl in crypts pretreated with CCh/THPS. These findings suggest that, in the absence of extracellular Ca 2+, release from intracellular stores is responsible for a component of the fura 2 signal achieved during exposure to NH 2 Cl. In further studies, we first depleted the intracellular stores by exposing crypts to CCh/THPS and then monitored the response to NH 2 Cl in the presence or absence of TPEN. As also shown in Fig. 8, the response to NH 2 Cl was nearly abolished when TPEN was present. These findings indicate that, after elimination of extracellular Ca 2+, the fura 2 signal in response to NH 2 Cl can be attributed largely to release from THPS-sensitive intracellular Ca 2+ stores and intracellular pools of heavy metal cations that are sensitive to TPEN.


Fig. 8. Fura 2 signals were monitored during exposure to 200 µM NH 2 Cl 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-NH 2 Cl baseline. P < 0.05 vs. the response to that observed under control conditions.


Studies were performed to more fully characterize NH 2 Cl-induced disturbances in Ca 2+ homeostasis, using 2-APB, a recognized inhibitor of store-operated channels. It has been reported that, under some experimental conditions, 2-APB may inhibit inositol trisphosphate receptor (InsP 3 ) actions, including release of intracellular stores. When such stores have been depleted, however, 2-APB has also been shown to block capacitative entry, independent of its effects on InsP 3 receptors ( 23, 43, 61 ). In these studies, fura 2-loaded crypts were exposed to NH 2 Cl (200 µM) in Ca 2+ -free Ringer containing TPEN (20 µM) to monitor an uncontaminated Ca 2+ signal. After NH 2 Cl-induced release of Ca 2+ from intracellular stores, crypts were exposed to Ca 2+ -Ringer (containing 20 µM free TPEN) alone or, in addition, 100 µM 2-APB. A representative recording is shown in Fig. 9 A, comparing responses of glands from the same harvest. In both recordings, peak effects and time course in response to NH 2 Cl (in Ca 2+ -free Ringer) and restoration of extracellular Ca 2+ were similar to those observed previously in the presence of TPEN ( Fig. 6 ). In the presence of 2-APB, added after store depletion by NH 2 Cl, the increases in i were significant ( n = 7 crypts in control and 2-APB groups).


Fig. 9. Effects of 2-aminoethoxydiphenylborate (2-APB) on store-operated entry induced by NH 2 Cl. Top : superimposed recordings from fura 2-loaded crypts from the same harvest. Both crypts were exposed to 200 µM NH 2 Cl, 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 NH 2 Cl, glands were exposed to 2-APB or vehicle (ethanol 1:1,000), and then extracellular Ca 2+ was restored. In the presence of 2-APB, increases in intracellular i; ratio) were diminished significantly; n = 7 crypts in each group. * P < 0.05.


Measurements of intracellular Zn 2+ concentration in isolated colon crypts using fluozin 3. To more conclusively identify the TPEN-sensitive component of the divalent cation signal, we performed studies in isolated crypts loaded with a recently reported Zn 2+ -sensitive fluorophore, fluozin 3 ( 27 ). This fluorophore has high affinity for Zn 2+ (reported K d 10-20 nM) and little expected interference from physiological concentrations of Ca 2+ ( 20, 64 ). Upon excitation, as shown in Fig. 10 A, loading appears similar to that observed with fura 2. However, little has been determined about the characteristics of fluozin 3 loading. To verify that the majority of the dye was loading in the cytoplasm and not organelles, experiments similar to those performed on fura 2-loaded crypts were undertaken to monitor cytoplasmic loss of dye during exposure to digitonin. In four separate experiments, crypts were loaded with 5 µM fluozin 3 and 5 µM magfura 2 simultaneously (data not shown). Fluorescence signals were monitored from both reporters by exciting fluozin 3 at 488 nm and magfura 2 at 340/380 nm. In these studies, the simultaneous presence of Magfura 2 ensured permeabilization ( 36 ) and allowed us to compare the rates at which both reporters were lost during the permeabilization. Crypts were perfused with ICB containing 5 nM free Zn 2+ and 40 µM pyrithione to increase the baseline signal intensity of fluozin 3. Digitonin (10 µM) was then added to induce the leakage of cytoplasmic dye. In each case, the intensity of fluozin 3 decreased by at least 85% after addition of digitonin, and the decline rates of signals were not visibly different (data not shown).


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 Zn 2+ buffered with EGTA in 0 Ca 2+ Ringer with 40 µM sodium pyrithione. Fluorescence is expressed in arbitrary units of intensity normalized to starting values (F/F o ). C : summary of calibrations in 6 individual crypts. With the use of averaged values in C, K d of fluozin 3 for Zn 2+ is 4 ± 0.3 nM.


In preliminary studies ( n = 4), we observed that, when crypts were exposed to Ringer solutions containing ionomycin (10 µM), TPEN (20 µM), and i ). Based on these studies, we calculate that in situ the K d of fluozin 3 for Zn 2+ is 4 ± 0.3 nM.


Effects of NH 2 Cl on i may increase to as much as 4-6 nM during exposure to 200 µM NH 2 Cl.


Fig. 11. Measurements of fluozin 3 signals during exposure of colon crypts to NH 2 Cl. A : recording in an individual crypt exposed to 200 µM NH 2 Cl in 0 Ca 2+ Ringer. Recording begins with crypts perfused by standard Ringer solution, then removal of Ca 2+, followed by exposure to NH 2 Cl (5 min), and recovery in 0 Ca 2+ Ringer. Sodium pyrithione (Na-pyr, 40 µM) was added in the absence of Zn 2+ followed by the addition of 1 µM Zn 2+. 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/F 0 ). Data represent recordings before (open bars), during (black bars), and after (shaded bars) removal of NH 2 Cl. * P < 0.05 compared with treatment before NH 2 Cl.


Effects of NH 2 Cl reactants, membrane-impermeable chloramines, and other control studies of reagents. In a series of control studies, we monitored fura 2 and fluozin 3 signals in isolated crypts during exposure to NH 3 (20 mM) and HOCl (200 µM), the reactants used to produce NH 2 Cl. At these concentrations, no changes in signal were observed for either NH 3 ( n = 4) or HOCl ( n = 4), indicating that observed effects were the result of NH 2 Cl and not its reactants (data not shown). Similarly, crypts were exposed to 200 µM taurine-NH 2 Cl, a stable, but membrane-impermeant, chloramine species ( 24, 62 ). No alterations were observed in fura 2 or fluozin 3 signals (data not shown), indicating that the effects of NH 2 Cl on i homeostasis are the result of its permeability in the cell membrane.


Additional control studies were performed to evaluate the possibility that NH 2 Cl 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 Ca 2+ and 20 µM fura 2 or 2 nM Zn 2+ and 20 µM fluozin 3. Addition of NH 2 Cl, HOCl, or NH 3 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 Zn 2+ and that Ca 2+ 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 Ca 2+ and Zn 2+ are not observed uniformly in response to all thiol oxidants.


Fig. 12. Fura 2 fluorescence recording from an individual fura 2-loaded crypt exposed to the nitric oxide donor S -nitroso- N -acetylpenicillamine (SNAP).


Intracellular thiols as targets of NH 2 Cl oxidation. We performed studies to confirm that NH 2 Cl-induced alterations in Ca 2+ and Zn 2+ homeostasis were because of release from thiol clusters that have been associated with proteins that bind or transport divalent cations ( 22, 42, 46, 48, 49, 59, 77 ). In one set of studies ( n = 4), we pretreated crypts with DTT (1 mM), a known sulfhydryl reducing agent ( 47, 62 ), before exposure to 200 µM NH 2 Cl. As shown in Fig. 13, as long as DTT was present in the perfusate, exposure to NH 2 Cl did not elicit alterations in fura 2 signals. When DTT was removed, however, responses to NH 2 Cl were immediately apparent. In other studies ( n = 4), DTT reversed the effects of NH 2 Cl when it was added after a peak effect was seen during NH 2 Cl exposure. To determine whether the presence of DTT might alter the concentration of NH 2 Cl in solution, we mixed 200 µM NH 2 Cl and 1 mM DTT in a Ringer solution in a cell-free chamber. Measuring NH 2 Cl absorbance at 242 nm, we found that the concentration of NH 2 Cl did not decrease in the presence of DTT, indicating that the effects DTT were not attributable to consumption of NH 2 Cl.


Fig. 13. Thiol reduction as a means of preventing or reversing NH 2 Cl effects. A : recording in an individual crypt exposed to 200 µM NH 2 Cl in 0 Ca 2+ Ringer already containing 1 mM dithiothreitol (DTT). DTT was then removed with NH 2 Cl remaining present. The crypt was allowed to recover in 0 Ca 2+ 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 NH 2 Cl in 0 Ca 2+ Ringer. NH 2 Cl was later removed with DTT remaining present. Again, the crypt was allowed to recover in 0 Ca 2+ Ringer, followed by addition of standard Ringer.


In an additional set of control studies, we monitored the ability of DTT to chelate Zn 2+ or Ca 2+. Using fluozin 3 ( K d for Zn 2+ 3 nM) and fluozin 2 ( K d for Zn 2+ 1-2 µM; see Ref. 27 ), we monitored fluorescence (excitation 495 nm, emission 520 nm) of both reporters in cell-free KCl solutions (150 mM) containing standard total concentrations of Zn 2+ using a 96-well plate fluorimeter. When fluozin 3 was present as the reporter, concentrations were calculated (maxchelator) to provide free Zn 2+ concentration ( i in the nanomolar range. In similar cell-free studies, the influence of DTT on Ca 2+ content of solutions was negligible, indicating that observed effects of DTT on NH 2 Cl-induced signals were not attributable to chelation of divalent cations.


DISCUSSION


To our knowledge, measurements of 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 NH 2 Cl causes dose-dependent increases in 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 NH 2 Cl ( 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 NH 2 Cl leads to dose-dependent increases in i. These findings offer the possibility that there is a range of concentrations over which NH 2 Cl 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. Ca 2+ -sensing fluorescent indicator dyes all are responsive to other polyvalent cations. Some potentially interfering ions (such as Cd 2+, Ba 2+, and La 3+ ) are excitatory (i.e., elicit responses similar to Ca 2+ ) but are not expected to interfere because they can only be added exogenously. In addition, some toxic cations (Mn 2+, Cd 2+, and Pb 2+ ) cause quenching or shifts in excitation spectra, rather than amplification of intensities; thus, they do not elicit responses similar to those of Ca 2+. Potentially interfering divalent cations include Fe 2+, Fe 3+, Cu 2+, and Zn 2+, which might be released from intracellular pools. The most widely used fluorescent reporter, fura 2, is quenched in response to Cu 2+, Fe 2+, and Fe 3+, but it responds to Zn 2+ in a similar manner to Ca 2+ ( 38 ). In fact, previous reports and manufacturer specifications indicate that, in vitro, fura 2 is more responsive to Zn 2+ ( K d 3-15 nM) than it is to Ca 2+ ( K d 150-300 nM; see Refs. 27, 38, and 54 ). Our calibration studies confirm these ranges of sensitivities to Ca 2+ and Zn 2+ in situ in cells of the rat colon crypt. These considerations suggest that, when fura 2 is used to monitor changes in , signals may include intracellular accumulation of Zn 2+ in the nanomolar range.


Our studies demonstrate that fura 2 monitors increases in 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 NH 2 Cl causes accumulation of Ca 2+ and Zn 2+ within the cytoplasm of the colon crypt cell. With respect to i, implying that THPS-sensitive stores are the major intracellular sources for the Ca 2+ signal. These responses are attributable to thiol modification of Ca 2+ transport processes, since DTT prevented and reversed effects of NH 2 Cl on fura 2 signals.


The molecular basis for oxidant-induced release of Ca 2+ from intracellular stores is not clear. In addition to direct modification of Ca 2+ release channels in the membrane of the endoplasmic reticulum ( 16, 77 ), thiol oxidants might alter receptors regulating release of Ca 2+ from stores to the cytoplasm. In this regard, both InsP 3 receptors ( 44 ) and ryanodine receptors ( 37 ) have been identified in colon crypts, the former having also been shown to support Ca 2+ -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 NH 2 Cl 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 Ca 2+, however, make it clear that entry of extracellular Ca 2+ is responsible for a major component of the NH 2 Cl-induced signal ( Figs. 6 and 7 ). Entry of extracellular Ca 2+ might be because of influx through capacitative entry, activated when CCh- and THPS-sensitive stores are released (29-31). Alternatively, NH 2 Cl-induced entry of extracellular Ca 2+ might be independent of store emptying. In the present study, we found that 2-APB inhibits a significant component of NH 2 Cl-stimulated entry of extracellular Ca 2+, arguing that NH 2 Cl 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 NH 2 Cl-induced store emptying and influx of extracellular Ca 2+. It is possible that exposure to NH 2 Cl 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 NH 2 Cl might modify Ca 2+ 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 Ca 2+ supplies a major component of the sustained increase in i caused by NH 2 Cl. It seems likely that the activation of such influx is at least partially independent of the effects of NH 2 Cl on emptying of intracellular Ca 2+ stores and is the result of thiol modification.


With respect to increases in in colon crypts under physiological conditions and during oxidative stress. In addition, they provide evidence that NH 2 Cl, and other oxidants that preferentially target S-H groups, may be highly effective in releasing Zn 2+ to the cytoplasm of cells in the colon crypt.


The final issue raised by our studies involves the consequences of oxidant-induced increases in 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 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 NH 2 Cl in injury and protection in acute colitis.


GRANTS


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).

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