Am J Physiol Cell Physiol AJP: Gastrointestinal and Liver Physiology
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Am J Physiol Cell Physiol 292: C1906-C1914, 2007. First published January 10, 2007; doi:10.1152/ajpcell.00550.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

Aliye Uc,1 Krzysztof J. Reszka,5 Garry R. Buettner,3 and John B. Stokes2,4

Departments of 1Pediatrics, 2Internal Medicine, and 3Radiation Oncology, 4Veterans Administration Medical Center and The University of Iowa, Carver College of Medicine, Iowa City, Iowa; and 5Department of Internal Medicine, Veterans Affairs Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 27 October 2006 ; accepted in final form 6 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heme induces Cl– secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl– secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents (Isc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced Isc was caused by Cl– secretion because it was inhibited in Cl–-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl– secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced Isc. SnPP-induced Isc was inhibited by the antioxidant vitamins, {alpha}-tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl– secretion can be regulated by the redox environment of the cell.

heme oxygenase; electrolyte transport; carbon monoxide; cGMP; reactive oxygen species

INTESTINAL EPITHELIAL CELLS maintain fluid-electrolyte homeostasis by keeping a tight barrier and regulating ion channels (9, 32). Many factors, including microbial agents, toxins, drugs, or inflammation, can disrupt this intestinal barrier and induce secondary messengers such as cAMP and cGMP (15, 17, 33). These secondary messengers can modulate intestinal fluid-electrolyte balance by inducing epithelial Cl– secretion (22, 42). We (45) have recently shown that heme and one of its degradation products, carbon monoxide (CO), can induce Cl– secretion in intestinal epithelial cells via cGMP formation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO) (35).

Heme prosthetic groups are found ubiquitously in living organisms (hemoglobin, myoglobin, catalase, peroxidases, cytochrome c, guanylate cyclase, and nitric oxide synthase), and they participate in important physiological functions (34). Despite its vital functions, free heme can be a source of ROS and promotes several deleterious cellular processes (4, 18). Intracellular heme levels are tightly controlled by adjusting a fine balance between its biosynthesis and catabolism (34). Heme is degraded by HO with the formation of CO and biliverdin and the release of iron from the porphyrin ring (43). The HO enzyme exists in two major forms: HO-1 and HO-2 (1, 28). HO-1 protein is inducible by numerous stimuli including heavy metals, heme, cytokines, hypoxia, or heat shock; HO-2 is a constitutively synthesized enzyme (27).

Because our previous work (45) showed that heme and CO caused electrolyte secretion in intestinal epithelial cells, we hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP) (12, 35), would inhibit heme-induced Cl– secretion. Contrary to our expectations, SnPP did not inhibit the short-circuit current (Isc) induced by hemin. SnPP caused a higher rate of Cl– secretion than hemin itself, an effect that was light dependent and inhibited by antioxidants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue culture. Caco-2 cells were kindly provided by Dr. Jeffrey Field (University of Iowa, Iowa City, IA) and used between passages 30 and 55. Stock cultures were grown to confluency at 37°C in 10% CO2 using DMEM (Mediatech, Cellgro, Herndon, VA) containing 4.5 g/l glucose, 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 15 mM HEPES. T84 cells were obtained from the American Tissue Culture Collection and grown in a 1:1 mixture of DMEM and Ham's F-12 media, 5% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM NaHCO3, and 15 mM HEPES. Fresh medium was added every 2 days. Cells were released from stock plates by a brief trypsin-EDTA treatment and plated at 30,000 cells/cm2 on Millipore Millicell PCF filters (Millipore, Billerica, MA). Development of a confluent monolayer was confirmed when transepithelial resistance (TER) was stable at ~1,300 {Omega}·cm2 for 2 successive days (about 14–16 days after cells had been seeded).

Electrical measurements. Transmonolayer voltage, TER, and Isc were measured in Ussing chambers constructed to accommodate Millicell filters (Jim's Instruments, Iowa City, IA) (23). For these measurements, cells were bathed in a Krebs-Ringer-bicarbonate solution consisting of (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 5 Na-HEPES, 5 H-HEPES, 1.5 CaCl2, 1 MgCl2, 1 Na2HPO4, and 5 glucose. The Cl–- and HCO3–-free Na+-HEPES solution contained (in mM) 1.5 CaNO3, 5 K-gluconate, 1 MgSO4·7H2O, 1 Na2HPO4, 5 Na-HEPES, 5 H-HEPES, 140 Na-isethionate, and 5 glucose. The Krebs-Ringer-bicarbonate solution was gassed with 5% CO2 at 37°C to maintain pH at 7.4; the Cl–- and HCO3–-free Na+-HEPES solution was gassed with air. A positive Isc value represents a flow of positive charge from the luminal (apical) to the basolateral solution (absorption) or a flow of a negative charge from the basolateral solution to the apical solution (secretion).

Cell viability assay. Cell viability was determined by the 3-[4,5-demethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. This assay relies on the production of formazan by the action of mitochondrial enzymes on MTT by living cells and correlates well with other measures of cell numbers (19). Confluent Caco-2 cells were treated with SnPP diluted in phenol red-free MEM (GIBCO-BRL Invitrogen, Carlsbad, CA) with L-glutamine in the apical or basolateral solution for 24 h at 37°C and 10% CO2. At the end of the treatment, media containing SnPP were aspirated, and cells were maintained in Krebs-Ringer-bicarbonate solution. Cells were then exposed to ambient fluorescent light in the laboratory at room temperature for 30 min, 1 h, and 2 h. Following light exposure, cells were incubated with MTT (0.5 mg/ml, Sigma Aldrich, St. Louis, MO) at 37°C and 10% CO2 for 2 h. The formazan formed was solubilized in 2-propanol and quantitated by measuring the absorbance at 550 nm.

Measurement of cell-associated SnPP. The SnPP content of Caco-2 cells was determined using the protocol previously described (4, 46). Media containing SnPP were aspirated, and cells were washed with HBSS three times. Cells were removed with 2 x 0.5 ml formic acid washes. The SnPP content of cells was determined spectrophotometrically (extinction coefficient at 409 nm = 2.8 x 105 M–1·cm–1) using a diode array UV/visible spectrophotometer (Agilent Technology, Palo Alto, CA). Cell-associated SnPP was expressed as nanomoles per filter over time.

Measurement of intracellular SnPP with confocal microscopy. We took advantage of SnPP's endogenous fluorescence and measured SnPP uptake by using confocal laser microscopy (Zeiss, Central Microscopy Facility, University of Iowa) and setting excitation at 543 nm and the emission at >560 nm (49). To help localize SnPP inside the cells, another fluorescent dye, To-Pro-3, was used to stain nuclear DNA (41).

Electron paramagnetic resonance measurements. Caco-2 cells were suspended in PBS (100,000 cells/ml) and transferred into a TM-flat cell for electron paramagnetic resonance (EPR) measurements. All EPR spectra were obtained at room temperature with a Bruker EMX spectrometer operating at 9.76 GHz. The EPR spectrometer settings were as follows: modulation frequency, 100 kHz; modulation amplitude, 1.0 G; microwave power, 40 mW; and receiver gain, 105–106. EPR spectra were recorded while the sample was being exposed to visible light (tungsten lamp) in the EPR cavity.

Chemicals. All chemicals were obtained from Sigma Aldrich unless otherwise stated. CFTR(inh)-72 (2) was a generous gift from Dr. Jonathan D. Kaunitz. SnPP was purchased from Frontier Scientific (Logan, UT) and diluted in 0.1 N NaOH. To-Pro-3 was purchased from Molecular Probes (Carlsbad, CA). Hemin (3 mM, Sigma-Aldrich) was prepared in 1 M NaOH and diluted to 0.5 mM in cell culture media and 10% FCS. The pH was adjusted to 7.4 by the slow addition of 12 M HCl. For convenience, we use heme (Fe2+) and hemin (Fe3+) interchangeably.

Statistics. Within-subject analyses (i.e., comparing Isc at baseline and after intervention) were performed using paired-sample t-tests. Between-subject analyses (i.e., Isc between control and SnPP groups) were performed using two-sample t-tests. Results are expressed as means ± SE. Statistical significance was defined as P < 0.05 (two-tailed analysis).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SnPP induces Isc in Caco-2 cells. Because heme and the product of heme degradation CO can induce Cl– secretion by Caco-2 cells (45), we hypothesized that a competitive inhibitor of HO, SnPP (12, 13), would inhibit heme-induced Cl– secretion. To test this hypothesis, Caco-2 cells were treated with hemin (100 µM), SnPP (50 µM), or hemin plus SnPP from the basolateral side of Caco-2 cells for 24 h. Baseline Isc was 1.5-fold higher in hemin-treated cells (Fig. 1, A and B). Contrary to our expectations, SnPP did not inhibit the Isc increase caused by hemin. Interestingly, SnPP treatment alone increased Isc gradually in Caco-2 cells. Cotreatment of SnPP with hemin inhibited the current induced by SnPP.


Figure 1
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  		Fig. 1. Tin protoporphyrin (SnPP) induces short-circuit current (Isc) in Caco-2 cells. A: confluent monolayers were treated with SnPP (50 µM), hemin (100 µM), or SnPP + hemin in the basolateral solution for 24 h. Isc was measured in Ussing chambers. Baseline Isc was higher in hemin-treated cells; Isc increased gradually in SnPP treated cells. Cotreatment of SnPP and hemin inhibited the current induced by SnPP. B: summary of Isc values. *P < 0.05 and **P < 0.01 compared with control; n = 9.

 
Basolateral but not apical SnPP induces Isc. We (46) have previously shown that cells exposed to hemin from the basolateral surface demonstrate a higher HO-1 induction than cells exposed to hemin from the apical surface. In addition, hemin increases Isc only if applied from the basolateral surface (45). To investigate if Caco-2 cells have a similar response to SnPP, we treated the cells with apical or basolateral SnPP (50 µM) for 24 h. Cells were then mounted in Ussing chambers and bathed with Krebs-Ringer solution that did not contain SnPP. Apical SnPP did not increase Isc (Fig. 2). In contrast, basolateral SnPP increased Isc significantly.


Figure 2
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  		Fig. 2. Effect of SnPP on Isc. Caco-2 cells were treated with SnPP (50 µM) in the apical or basolateral solution for 24 h. Apical SnPP had a minimal effect on Isc [P = not significant (NS) compared with control; n = 6], whereas basolateral SnPP greatly enhanced Isc (*P < 0.01 compared with control; n = 6).

 
Cell-associated SnPP is higher following basolateral exposure. We postulated that the greater Isc response to basolateral SnPP was related to SnPP having better access to the Caco-2 cell cytosol from this side. To test this hypothesis, we measured cell-associated SnPP levels in Caco-2 cells (4, 46). Confluent Caco-2 cells were treated with SnPP (5–50 µM) in the apical or basolateral solution for 24 h. Cell-associated SnPP was much higher after basolateral treatment compared with apical treatment at all concentrations (Fig. 3).


Figure 3
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  		Fig. 3. Cell-associated SnPP. Caco-2 cells were treated with 5–50 µM SnPP from the apical or basolateral side for 24 h. Cells were removed with formic acid, and cellular SnPP content was measured spectrophotometrically. Cell-associated SnPP was much higher after basolateral exposure. *P < 0.01, apical vs. basolateral; n = 4.

 
We also assessed intracellular localization with confocal laser microscopy. The SnPP signal inside the cell was barely detectable after apical SnPP application (data not shown). In contrast, after basolateral treatment, the fluorescent SnPP signal was abundant in the cytoplasm; the signal from the nucleus was minimal (Fig. 4). These results indicate that the greater cell-associated SnPP following basolateral exposure is due to cell uptake of SnPP and not to extracellular binding. The results suggest that SnPP must gain access to an intracellular pool to produce its effects on Isc.


Figure 4
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  		Fig. 4. Intracellular SnPP localization. Caco-2 cells were treated with 50 µM SnPP for 2 h and counterstained with To-Pro-3 after being fixed with 2% paraformaldehyde. SnPP was visualized by setting excitation at 543 nm and emission at >560 nm (red color); To-Pro-3 was visualized by setting the excitation at 633 nm and emission at 650 nm (blue color). SnPP was diffusely distributed inside the cells and did not localize to the nucleus. This image is representative of 3 different experiments.

 
SnPP dose response in Caco-2 cells. To determine the effect of concentration on SnPP-induced Isc in Caco-2 cells, we treated cells with SnPP (10, 25, and 50 µM) for 24 h and analyzed Isc responses in Ussing chambers (Fig. 5, A and B). Maximum Isc was reached much faster with 50 µM SnPP compared with 10 and 25 µM SnPP (66.2 ± 4 min for 10 µM, 65.9 ± 4.2 min for 25 µM, and 42.9 ± 5.8 min for 50 µM; P < 0.01, 50 vs. 10 or 25 µM SnPP), but peak current was not as great as that recorded from cells exposed to 10 or 25 µM SnPP. To determine if cell toxicity played a role in this response, we performed a MTT assay on cells treated with 50 µM SnPP for 24 h (basolateral) at 0, 10, 40, and 100 min (the time point when Isc had declined) after starting the experiment. Evidence of cell toxicity was observed only at 100 min. The MTT assay was also done after cells were treated with 10 µM basolateral SnPP for 24 h and kept in Ussing chambers for similar time points; no cell toxicity was seen at any time point examined. Therefore, it is possible that the relatively lower peak Isc response to 50 µM SnPP is the result of some cell toxicity.


Figure 5
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  		Fig. 5. SnPP dose response. A: representative time course. Caco-2 cells were treated with 10, 25, and 50 µM SnPP in the basolateral solution for 24 h. Isc was lowest with 50 µM SnPP; the time to reach maximum Isc was longest with 10 µM SnPP. B: summary of peak Isc data. *P < 0.01, control vs. treatment; n = 7.

 
SnPP dose response in T84 cells. To determine if the results were unique to Caco-2 cells, we examined the responses to SnPP in another intestinal epithelial cell line, T84 cells. Cells were treated with SnPP (10, 25, and 50 µM) for 24 h (Fig. 6, A and B). In contrast to Caco-2 cells, SnPP induced Isc in a dose-dependent way in T84 cells. SnPP at 50 µM produced a consistent stimulation of Isc, whereas 10 µM produced almost no stimulation and 25 µM had an intermediate effect. No cell toxicity was observed with SnPP at these concentrations at any time point, suggesting that T84 cells were more resistant to higher concentrations of SnPP than Caco-2 cells.


Figure 6
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  		Fig. 6. SnPP dose response in T84 cells. A: representative time course of monolayers treated with 10, 25, and 50 µM SnPP in the basolateral solution for 24 h. The Isc response to SnPP was dose dependent. B: summary of peak Isc data. *P < 0.01 compared with control; n = 5.

 
SnPP-induced Isc is Cl– secretion. To determine whether Na+ absorption via the epithelial Na+ channel (ENaC) could account for the SnPP-induced Isc, we treated the cells with benzamil (10 µM) (29) in the apical solution of Ussing chambers. Benzamil had no effect on Isc, suggesting that Na+ absorption via ENaC did not have a role in the generation of this current (Fig. 7).


Figure 7
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  		Fig. 7. SnPP-induced Isc is not caused by electrogenic Na+ absorption. Caco-2 monolayers were treated with 50 µM SnPP for 24 h. The epithelial Na+ channel inhibitor benzamil (10 µM) had no effect on SnPP-induced Isc. *P < 0.01, control vs. SnPP; P = NS, SnPP vs. SnPP + benzamil; n = 7.

 
To determine the nature of the Isc induced by basolateral SnPP exposure, Ussing chamber experiments were repeated in Cl–- and HCO3–-free Na+-HEPES solution. The SnPP-induced current was significantly reduced if Cl– and HCO3– were absent in Ussing chambers, suggesting that the Isc increase caused by SnPP was due mostly to active Cl– secretion (Fig. 8).


Figure 8
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  		Fig. 8. SnPP-induced Isc is Cl– dependent. Caco-2 cells were treated with basolateral SnPP (50 µM) for 24 h. SnPP-induced Isc was significantly lower if Cl– and HCO3– were absent. *P < 0.01, control vs. SnPP; **P < 0.01, SnPP in Krebs-Ringer solution vs. SnPP in Cl–- and HCO3–-free HEPES; n = 4.

 
The nature of SnPP-induced Isc was further examined by ouabain, an inhibitor of basolateral Na+-K+-ATPase, a pump needed to maintain low intracellular Na+ levels (37). Ouabain inhibited SnPP-induced Isc (Table 1), indicating that this stimulated current was dependent on the Na+-K+ pump. These results imply that this current does not represent an active transport process powered by a H+-ATPase. We then tested the effect of a Cl– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (5), on SnPP-induced Isc. NPPB (100 µM) applied to the apical surface of the cells significantly inhibited Isc caused by SnPP (Table 1). These results indicate that SnPP-induced Isc was the result of active Cl– secretion and not Na+ absorption.


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  		Table 1. SnPP-induced Isc is CI– dependent

 
To investigate if the current induced by SnPP was caused by cGMP or cAMP, we used inhibitors. After being treated with SnPP (10 µM basolateral) for 24 h, cells were incubated in Ussing chambers with the soluble guanylate cyclase inhibitor [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 25 µM) or inhibitors of cAMP-dependent protein kinase Rp-cAMPS (50 µM) and H-89 (10 µM). Neither agent had any effect on SnPP-induced Isc, suggesting that this current was not dependent on cAMP or cGMP.

The role of CFTR on SnPP-induced current was investigated by treating the monolayers with a potent and selective CFTR inhibitor, CFTR(inh)-72 (2). We observed no change in SnPP-related responses with CFTR(inh)-72 (2 µM). Therefore, it is unlikely that SnPP-induced Cl– secretion is caused by CFTR activation.

SnPP-induced Isc is light dependent. SnPP is an efficient photosensitizer that can generate singlet oxygen (1O2) after exposure to light (26). 1O2 can react with electron-rich moieties in lipids, amino acids, and nucleic acids, causing their oxidation (30). Because the SnPP effect required 45–60 min to develop, we thought that the photooxidation initiated by SnPP and ambient (fluorescent) light could be playing a role. To test this idea, we covered one control and one SnPP-treated (50 µM basolateral) set of Caco-2 cells with aluminum foil for the first 30 min of the experiment and then removed the covers (Fig. 9). Isc increased in SnPP-treated cells only after they were exposed to light; light had no effect on cells not treated with SnPP.


Figure 9
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  		Fig. 9. SnPP-induced Isc is light dependent. Caco-2 cells were treated with basolateral SnPP (50 µM) for 24 h. One control and one SnPP-treated well were covered with aluminum foil the first 30 min. Isc increased only after SnPP-treated cells were exposed to ambient fluorescent lights. The time course is representative of 5 different experiments.

 
To determine whether SnPP undergoes photodegradation during light exposure, we measured changes in its absorption spectrum. SnPP (10 µM) was diluted in the Krebs solution used to measure Isc, and absorption spectra of the solution were obtained at time points up to 7 h while the solution was continuously exposed to ambient fluorescent light. There was no change in SnPP absorption after 30 min of light exposure. Starting at 1 h, the absorption of SnPP (407 nm) decreased, and isosbestic points appeared at 398 and 418.5 nm (Fig. 10). These results indicate that SnPP underwent a chemical change upon light exposure.


Figure 10
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  		Fig. 10. Degradation of SnPP with ambient fluorescent light. SnPP was diluted in Krebs solution (10 µM), and spectra were obtained at 0 h, 30 min, and hourly for 7 h while SnPP was continuously exposed to ambient fluorescent light. SnPP absorption at 407 nm decreased over time with light exposure, and isosbestic points ({lambda}ISO) appeared at 398 and 418.5 nm. The results shown are representative of 3 separate experiments.

 
We then asked if SnPP that had been exposed to light in solution could reproduce the effects on Caco-2 cells acutely. First, we added freshly prepared SnPP to Ussing chambers (10 µM) from the basolateral surface. This treatment caused no increase in Isc, suggesting that a prolonged incubation and possibly intracellular accumulation were required to see an effect. Second, we prepared SnPP (10 µM) in Krebs solution and exposed to the ambient fluorescent light for 24 h. Caco-2 cells were then incubated with this solution in Ussing chambers from the basolateral surface. This treatment did not cause an increase in Isc. Therefore, the end product of SnPP degradation upon light exposure did not seem to reproduce the effect of light on cells previously exposed to SnPP. These results suggest that the stable degradation product of SnPP is not the agent responsible for increasing Cl– secretion. Thus, the action of light on intracellular SnPP must generate an intermediate product that induces Cl– secretion.

SnPP-induced Isc is caused by oxidative stress. Photooxidative effects of SnPP were initially described in animals who received this compound along with phototherapy (14, 21, 25). Further studies (10, 31) have suggested that the phototoxic reactions observed with SnPP were the results of lipid peroxidation. To examine the role of lipid peroxidation on SnPP-induced Isc, we examined the effects of {alpha}-tocopherol, an inhibitor of the free radical chain reactions of lipid peroxidation (7). Caco-2 cells were pretreated with {alpha}-tocopherol (10 µM) for 5 days prior to being treated with SnPP. {alpha}-Tocopherol significantly inhibited the effect of SnPP (Fig. 11, A and B), suggesting that lipid peroxidation was playing a role in Cl– secretion induced by SnPP.


Figure 11
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  		Fig. 11. {alpha}-Tocopherol inhibits SnPP-induced Isc. Caco-2 cells were treated with basolateral SnPP (50 µM) for 24 h. A: time course of the Isc response to SnPP. B: summary of the peak response to SnPP experiments: {alpha}-tocopherol inhibited SnPP-induced Isc. *P < 0.01, control vs. SnPP; **P < 0.05, SnPP vs. SnPP + {alpha}-tocopherol; n = 4.

 
We then studied the effects of another antioxidant, ascorbic acid (7). Ascorbate has been reported to protect SnPP-treated rats against phototoxicity (24). Ascorbic acid (1 mM) was given to Caco-2 cells following 24 h treatment with SnPP (10 µM basolateral) at the time of mounting the cells in the Ussing chambers. Ascorbate abolished the increase in current caused by SnPP (Fig. 12, A and B). These results further confirm that the effect of SnPP + light is mediated via oxidation reactions.


Figure 12
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  		Fig. 12. Ascorbic acid [vitamin C (Vit C)] abolishes SnPP-induced Isc. Caco-2 cells were treated with basolateral SnPP (10 µM) for 24 h. A: time course of the effect of Vit C (1 mM). B: summary of the peak response to SnPP. Vit C inhibited SnPP-induced Isc. *P < 0.01, control vs. SnPP; **P < 0.01, SnPP vs. SnPP + Vit C; n = 5.

 
To determine the role of oxidative stress in SnPP-induced Cl– secretion, we used EPR. We took advantage of ascorbate's ability to react with oxidants with the subsequent formation of the ascorbate free radical (Asc·–). The level of Asc·– is a real-time indicator of the flux of oxidants in a system (8). Caco-2 cells were treated with SnPP (10 µM) from the basolateral surface for 24 h and ascorbate (1 mM) for the last 2 h. Cells were then removed from the filters and analyzed by EPR. As expected, a background level of Asc·– was observed (Fig. 13A). However, upon exposure to light, the steady-state level of Asc·– was increased (Fig. 13B). These data support the idea that oxidative reactions can be initiated with light in a cell sample loaded with SnPP.


Figure 13
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  		Fig. 13. Light increased the electron paramagnetic resonance (EPR) signal intensity of the ascorbate radical (Asc·–). Caco-2 cells (100,000 cells/ml) were treated with SnPP (10 µM) basolaterally for 24 h and ascorbate (1 mM) for 2 h. Cells were examined by EPR. A: in the absence of light, a small Asc·– signal is seen, consistent with slow, background oxidation of ascorbate. B: visible light (tungsten lamp) increased the intensity of the Asc·– signal (n = 3). This increase was not seen in the absence of SnPP (data not shown). AU, arbitrary units.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we used SnPP to inhibit the effects of hemin on intestinal epithelial cell Cl– secretion. Contrary to our expectations, SnPP produced an increase in Isc. We found that the SnPP effect on Isc was light dependent and inhibited by antioxidants.

The Isc increase caused by SnPP was the result of active Cl– secretion: it was significantly inhibited by the absence of Cl– in Ussing chamber solutions. It was not inhibited by benzamil, a specific inhibitor of ENaC, but was inhibited by NPPB, a nonselective inhibitor of anion transporters. The inhibition by ouabain, an inhibitor of Na+-K+-ATPase, which functions to maintain low intracellular Na+ levels in cells (11), further supports the conclusion that the SnPP-stimulated Isc is Cl– secretion. A low intracellular Na+ concentration is necessary to allow the entry of Na+ and Cl– across the basolateral membrane. Cl– accumulated inside the cell is secreted across the apical membrane through apical Cl– channels. The inhibitory effect of ouabain on this current demonstrates that SnPP-induced Cl– secretion requires a Na+ gradient maintained by basolateral Na+-K+-ATPase. This effect of ouabain greatly reduces the possibility that this current could have been powered by an H+-ATPase.

Intestinal epithelial Cl– secretion is dependent on the activation of apical Cl– channels. One of the best-characterized apical Cl– channel is CFTR, a channel that is activated via cAMP (6). In our experiments, two cAMP inhibitors and a specific CFTR inhibitor had no effect on SnPP-induced current. Therefore, it is unlikely that SnPP induces Cl– secretion via CFTR and/or cAMP activation. On the other hand, SnPP-induced Isc was reduced by NPPB, a nonspecific Cl– transporter inhibitor. These results, taken together, suggest that the apical Cl– channel responsible for SnPP-stimulated Cl– secretion is not CFTR but another kind of Cl– channel.

SnPP stimulated Cl– secretion only if the cells were treated from the basolateral surface and not from the apical or lumen side. This differential effect was most likely the result of SnPP having better access to an intracellular compartment from the basolateral side. These observations, along with hemin (iron-protoporphyrin) inducing HO-1 more efficiently from the enterocyte basolateral surface (46), suggest that intestinal epithelial cells have a more efficient uptake and transport mechanisms for porphyrins from their basolateral membrane.

We observed that SnPP induced Cl– secretion only when cells were exposed to ambient light. One possibility we considered was the generation of CO from the light degradation of SnPP, as demonstrated by Vreman and colleagues (47). CO induces Cl– secretion in intestinal epithelial cells via cGMP, an effect we (45) have previously shown to be inhibited by the soluble guanylate cyclase inhibitor ODQ. However, CO generation was an unlikely intermediate of the SnPP-increased Isc because ODQ had no effect on the current.

We have determined that Cl– secretion in Caco-2 cells was due to oxidative events induced by SnPP and light. This deduction is based on the fact that the SnPP-induced current is inhibited by two antioxidants, lipid-soluble {alpha}-tocopherol (vitamin E) and water-soluble ascorbate (vitamin C). This conclusion is further supported by the observed increase in the steady-state level of Asc·– in cells that were exposed to light after being treated with SnPP.

Another observation consistent with the conclusion that the light-mediated SnPP effect is related to oxidation is that coincubation of hemin and SnPP reduced the effect of SnPP alone on Isc. Although hemin is capable of inducing Cl– secretion in intestinal epithelial cells (45), it reduced the Cl– secretory effects of SnPP. It seems possible that the inhibitory effect of hemin on SnPP-induced Isc was secondary to HO-1 induction. The inhibitory effect of hemin occurred after 24 h of treatment, a time point when HO-1 is induced in these cells (44). Degradation of heme via HO-1 generates antioxidant products such as bilirubin and biliverdin (38, 39). These molecules might have inhibited the SnPP response by changing the redox environment of the cell. An extension of this idea is that these metabolic products of heme might mitigate an otherwise strongly oxidative state induced by heme itself.

Another important conclusion from this study is the role of SnPP in creating oxidative stress in vivo. SnPP is widely used in animal and cell culture experiments to inhibit the activity of HO-1. Interestingly, ambient laboratory light is sufficient to initiate significant photooxidative reactions of SnPP in cell culture studies. Therefore, the effects observed with SnPP may not be entirely due to its inhibition of HO-1 but also the generation of photooxidative products by this porphyrin. It is not known if other metalloporphyrins, such as cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), would have photosensitizing effects similar to SnPP in this setting. In general, CoPP and ZnPP are considered to be ineffective photosensitizers (10, 36). The possible effects of these metalloporphyrins will be studied in the future.

Our data suggest that SnPP and light induce changes in the redox environment of the cell, which leads to Cl– secretion. There is precedence for a role of the cellular redox status in the regulation of a Cl– channel, CFTR. Reducing conditions increase the open state of CFTR; in contrast, oxidation of the CFTR decreases the open state of the channel (20). While it seems very unlikely that SnPP-induced Cl– secretion in Caco-2 cells is mediated via CFTR, it does seem likely that changing the redox environment of the cell is intimately involved in regulating this Cl– secretion.

These results underscore the important role that the redox environment of the cell may play in electrolyte secretion. Such a regulatory system may be responsible, in part, for the Cl– secretion (and thus diarrhea) observed with intestinal inflammation. Toxic ROS and other oxidants can be generated during intestinal inflammation from macrophages and monocytes (40, 48). These molecules might contribute to tissue injury (3) and play an important role in the diarrhea observed in inflammatory conditions (16).

In summary, SnPP, a competitive inhibitor of HO, caused Cl– secretion at a higher magnitude than hemin itself, an effect that was light dependent and inhibited by antioxidants. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. This study also underscores the concept that the redox environment of the cell may play a role in regulating Cl– secretion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-63135 (to A. Uc) and DK-52617 (to J. B. Stokes) as well as grants from the Department of Veteran's Affairs (to J. B. Stokes).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Jonathan Kaunitz for providing CFTR(inh)-172.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Uc, 2865 JPP, Children's Hospital of Iowa, 200 Hawkins Dr., Iowa City, IA 52242 (e-mail: aliye-uc{at}uiowa.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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