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J Physiol (2003), 551.1, pp. 207-217
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.041921
-stimulated production of superoxide in rabbit gastric parietal cells |
ABSTRACT |
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We have shown previously that the G protein-coupled production of superoxide anion (O2-) leads to closure of small-conductance Cl- channels (0.3-0.4 pS) in the basolateral membrane of rabbit parietal cells. In the present study, effects of interleukin-1
(Received 19 February 2003; accepted after revision 30 May 2003; first published online 18 June 2003)
Corresponding author H. Sakai: Department of Pharmaceutical Physiology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. Email: sakaih{at}ms.toyama-mpu.ac.jp
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INTRODUCTION |
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A small-conductance Cl- channel (0.3-0.4 pS) present in the basolateral membrane of rabbit and rat gastric parietal cells has a unique range of physiological roles. First, this Cl- channel has a housekeeping role through domination of the cell membrane potential (Sakai et al. 1992, 1996; Ikari et al. 1999). Although K+ and non-selective cation channels are present, these cation channels do not significantly contribute to the membrane potential in parietal cells (Sakai et al. 1989). Second, the Cl- channel is opened via the prostaglandin E2/nitric oxide (NO)/cGMP-dependent pathway (Sakai et al. 1995), and activation of the Cl- channel leads to protection of the parietal cells against ethanol-induced cytotoxicity (Sakai et al. 1998). Third, the Cl- channel is closed by the G protein-mediated intracellular production of superoxide anion (O2-) (Sakai & Takeguchi, 1994); block of the Cl- channel by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) leads to cytotoxicity in the parietal cells, strongly suggesting a role for the Cl- channel in O2- cytotoxicity (Sakai et al. 1998). However, to date no ligand and receptor mediating inhibition of this Cl- channel has been found.
We focused on interleukin-1 (IL-1) as a candidate for the ligand because of the following reports. Helicobacter pylori, the gastro-toxic bacterium, induced expression of IL-1 mRNA (Mai et al. 1991) and the production of IL-1 (Aihara et al. 1998) in human peripheral blood mononuclear cells. IL-1 stimulated the production of O2- in rat pulmonary microvascular smooth muscle cells (Boota et al. 1996), rat cultured hypothalamic astrocytes (Tolias et al. 1999) and calf neutrophils (Hagiwara et al. 2001). Overexpression of manganese superoxide dismutase in rat insulinoma cells provided complete protection against IL-1-induced cytotoxicity (Hohmeier et al. 1998). In isolated rat gastric parietal cells, IL-1 receptor mRNA was expressed (Schepp et al. 1998).
In the present study, we investigated the effect of IL-1 on the small-conductance Cl- channel in rabbit gastric parietal cells, and found that IL-1 closes the Cl- channel via the G protein-mediated Rho/Rho-kinase-dependent production of O2-. Some of these results have been published in abstract form (Sakai & Takeguchi, 2001).
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METHODS |
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Chemicals
Y-27632 was a generous gift from Welfide (Osaka, Japan). Collagenase was obtained from Wako Pure Chemical (Osaka, Japan). Pronase (Actinase E) was from Kaken Pharmaceutical (Tokyo, Japan). Recombinant rat IL-1 was from PeproTech EC (London, UK). Anti-rat IL-1 antibody, normal goat IgG and recombinant human IL-1 receptor antagonist (IL-1ra) were from R&D Systems (Minneapolis, MN, USA). Dihydrofluorescein diacetate (H2FDA) was from Molecular Probes (Eugene, OR, USA). Carbamylcholine chloride (carbachol) was from Sigma (St Louis, MO, USA). Fluvastatin was from LKT Laboratories (St Paul, MN, USA). Copper-zinc superoxide dismutase (Wakamoto Pharmaceutical, Tokyo, Japan), catalase (Biozyme Laboratories, Blaenavon, UK), GDPS (Wako Pure Chemical) and Clostridium botulinum C3 exoenzyme (BIOMOL Research Laboratories, Plymouth Meeting, PA, USA) were dissolved in appropriate solutions just before use.
Isolation of gastric glands
The procedures described below were performed in accordance with the guidelines presented by the Animal Care and Use Committee of Toyama Medical and Pharmaceutical University. Male Japanese White rabbits (weighing 2-3 kg) were killed by the intraperitoneal administration of an overdose of urethane (> 2 g kg-1). The stomach was removed and the gastric mucosa was digested with 170 units ml-1 of collagenase at 35 °C for 40 min. Isolated glands were further treated with 500 tyrosine units ml-1 Actinase E at 25 °C for 5 min. As a result of the additional digestion, parietal cells protruded from the base of glands and had no leaky connection between the intracellular canaliculi and the lumen (Sakai et al. 1992). The glands were suspended in a standard medium containing (mM): 132.4 NaCl, 5.4 KCl, 5 Na2HPO4, 1 NaH2PO4, 1.2 MgSO4, 1 CaCl2, 2 mg ml-1 bovine serum albumin, 2 mg ml-1 glucose and 10 Hepes (pH 7.35).
Preparation of the parietal cell-rich suspension
Isolated rabbit gastric glands were treated with 4000 tyrosine units ml-1 Actinase E at 35 °C for 50 min. Then, parietal cells in the suspension were separated by use of a continuous Percoll gradient, which was formed by centrifuging a mixture of Percoll (Amersham Biosciences, Uppsala, Sweden), the standard medium (pH 7.0) and 1.5 M NaCl solution (45:50:5 in v/v/v) at 23 000 g for 50 min. The fraction of parietal cells was further purified with a Beckmann J2-21M elutriator centrifuge. It consisted of 82-89 % parietal cells.
RNA isolation, RT-PCR and Northern blot analysis
Poly A+ RNA of rabbit gastric parietal cells was prepared from the total RNA (isolated from the parietal cell-rich suspension) by using Poly ATtract mRNA isolation system II (Promega, Madison, WI, USA). RT-PCR was carried out as described elsewhere (Sakai et al. 1999, 2001). Two sets of sense and antisense primers were used for PCR: (1) a set of primers based on the human IL-1 receptor sequence (nucleotide positions 241-257: 5'-CTTTGGTTTGTTCCTGC-3' and nucleotide positions 1137-1157: 5'-TCATAGGTCTTTCCATCTGAA-3'), and (2) a set of primers based on the rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequence (nucleotide positions 443-461: 5'-CATCCTGCACCACCAACTG-3' and nucleotide positions 916-935: 5'-TACCAGGAAATGAGCTTCAC-3'). The amplified products were sequenced and used for preparation of the 32P-labelled cDNA probes. For Northern blot analysis, poly A+ RNA of rabbit gastric parietal cells (2.5 µg) was separated on a 1 % agarose/formamide gel and transferred onto a nylon membrane (Zeta-probe GT, Bio-Rad). The membrane was hybridised with the 32P-labelled cDNA fragment of rabbit IL-1 receptor or GAPDH for 12 h at 65 °C in 250 mM NaH2PO4-Na2HPO4 (pH 7.2)-7 % SDS, washed in 40 mM NaH2PO4-Na2HPO4 (pH 7.2)-1 % SDS at 65 °C, and exposed to the Imaging Plate (Fuji Film) for 6 h (GAPDH) or 48 h (IL-1 receptor).
Patch-clamp recordings
Protruded parietal cells in gastric glands could be morphologically distinguished under a phase-contrast microscope. Whole-cell currents recorded from rabbit parietal cells in the isolated gastric glands were previously established to be due to the current through the basolateral membrane (Sakai et al. 1992). In some experiments, whole-cell recordings were made in singly isolated parietal cells. A List EPC-7 patch-clamp system (List Electronic, Darmstadt, Germany) was used for whole-cell recordings as described elsewhere (Sakai et al. 1992; Sakai & Takeguchi, 1993). Series resistance and capacitance were compensated electronically. The liquid junction potential between the pipette and bathing solutions was corrected using the pipette offset on the amplifier. The following extracellular bathing and intracellular pipette solutions were used. The 133K+-142Cl- bathing solution contained (mM): 133 KCl, 7 NaCl, 1 sodium aspartate, 1 MgCl2, 1 CaSO4, 0.1 ouabain and 10 Hepes. The 140choline+-146Cl- bathing solution contained (mM): 140 choline chloride, 2 MgCl2, 1 CaCl2, 0.1 ouabain and 10 Hepes. The 133K+-13Cl- pipette solution contained (mM): 133 potassium aspartate, 7 NaCl, 3 MgCl2, 0.062 CaSO4, 2 ATP, 0.1 EGTA and 10 Hepes (pCa 7). The 140choline+-146Cl- pipette solution contained (mM): 140 choline chloride, 3 MgCl2, 0.062 CaSO4, 2 ATP, 0.1 EGTA and 10 Hepes (pCa 7). All the bathing and pipette solutions were adjusted to pH 7.3 with KOH or Tris. The membrane potential was recorded in the current-clamp mode. To obtain the current-voltage relationship with the 140choline+-146Cl- bathing and the 140choline+-146Cl- pipette solutions, command pulses were superposed on the holding potential (0 to ±100 mV, voltage jump by ±20 mV, duration of 400 ms; Sakai et al. 1992; Sakai & Takeguchi, 1993). Whole-cell Cl- currents were recorded continuously with the 133K+-142Cl- bathing solution and the 133K+-13Cl- pipette solution at a holding potential of 0 mV, the zero-current potential for K+ and non-selective cation channels (Sakai et al. 1996). Experiments were performed at 35 °C. Whole-cell Cl- currents were read just before (I0 min) and 10 min after (I10 min) the addition of IL-1, and then the data was shown as 100 
(I10 min/I0 min) (Sakai & Takeguchi, 1994; Sakai et al. 1995).
Noise analysis of the whole-cell Cl- current
Variance noise analysis was performed as previously described (Sakai et al. 1992; Sakai & Takeguchi, 1993; Ikari et al. 1999). Briefly, whole-cell Cl- currents which were observed in the presence of 1 ng ml-1 IL-1 were low-pass filtered (1 kHz), and recorded on the tape recorder (SONY A-65). For the noise analysis of the Cl- current, the tape was replayed at the same speed and currents were high-pass filtered (0.3 Hz). The variance of the fluctuation of the whole-cell Cl- current was calculated (2-200 Hz) using a signal analyser (SM-2100A, Iwatsu Electric Co., Tokyo, Japan). The unit amplitude of the single Cl- channel current (i) and total number of Cl- channels in the basolateral membrane (N) can be estimated from the following equation under several assumptions as previously reported (Sigworth, 1980a,b):
2/I = i - I/N,
where I is the mean whole-cell current and 2 is the variance of the fluctuation, which was calculated with the signal analyser. In the present experiment, it was assumed that the Cl- current of the basolateral membrane of rabbit parietal cells represents a single population of channels because the power spectra of whole-cell Cl- current fluctuations contained only one Lorentzian component (Sakai et al. 1992, 1996; Sakai & Takeguchi, 1993). In addition, N was assumed to be unchanged during the recording period of the Cl- current, because the present Cl- channel showed no time- and voltage-dependent activation or inactivation (Sakai et al. 1992; Sakai & Takeguchi, 1993).
Measurement of the production of intracellular oxygen radicals in single parietal cells
Isolated gastric glands were suspended in the BSA-free standard medium. Dihydrofluorescein diacetate (H2FDA; 5 µM) was added to the suspension and incubated for 30 min at 22 °C. After loading, the glands were washed and suspended in ice-cold standard medium. Then, they were warmed at 35 °C in the BSA-free standard medium before measurement. The H2FDA-loaded single parietal cells in gastric glands were observed under an inverted microscope. Dihydrofluorescein is non-fluorescent, whereas its oxidised form, fluorescein, is fluorescent (Hempel et al. 1999). The total fluorescence intensity of fluorescein from one single parietal cell was monitored at excitation wavelengths of 490 nm with an emission wavelength of 510 nm (interference filter) using a photon-counting technique (Spex Fluorolog-2 spectrofluorometer, Edison, NJ, USA).
Measurement of [Ca2+]i in single parietal cells
Fura-2/penta-acetoxymethyl ester (5 µM) and detergent, Pluronic F127 (0.025 %, w/v), were added to the suspension of isolated gastric glands and incubated for 30 min at 22 °C. The total fluorescence intensity of a Fura-2-loaded single parietal cell was monitored at 505 nm (interference filter) using a photon-counting technique (Spex Fluorolog-2 spectrofluorometer) following excitation at wavelengths of 340 and 380 nm. After corrections for background fluorescence, the intensity ratio (340/380 nm) and [Ca2+]i were calculated as described previously (Grynkiewicz et al. 1985).
Assay for Rho activation
Effects of IL-1 on Rho activity in purified rabbit gastric parietal cells (2 106 cells) were assessed using the Rho activation assay kit (Upstate, Lake Placid, NY, USA). The principles of this assay are based on the selective affinity precipitation of Rho-GTP with immobilised Rhotekin (Rho binding domain), and detection of Rho by Western blot with anti-Rho (-A, -B, -C) antibody. The blots were stained with the ECL Plus Western blotting kit (Amersham Biosciences, Buckinghamshire, UK).
Statistics
Results are presented as the means ± S.E.M. Differences between groups were analysed by one-way ANOVA, and correction for multiple comparisons was made by using Dunnett's multiple comparison test. If necessary, Tukey's multiple comparison test was used. Comparison between the two groups was made by using Student's t test. Statistically significant differences were assumed at P < 0.05.
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RESULTS |
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Inhibitory effect of IL-1 on the whole-cell Cl- current
We have previously shown that the whole-cell Cl- current of a rabbit gastric parietal cell arises from opening of one kind of basolateral small-conductance (sub-pS) Cl- channel (Sakai et al. 1992, 1996; Sakai & Takeguchi, 1993). In the present study, closure of the Cl- channel is observed as a decrease in the whole-cell Cl- current. Figure 1A shows that IL-1 (0.3-10 ng ml-1) inhibited the whole-cell Cl- current with a slow time course. The steady-state effect was observed about 10 min after the addition of IL-1. The maximal inhibitory effect was observed using 1-3 ng ml-1 of IL-1, and the effect was smaller at a higher concentration of 10 ng ml-1 (Fig. 1B). A similar concentration-response relation was also obtained with IL-1-stimulated invasion of human articular cartilage by rheumatoid synovial fibroblasts; the maximal effect was obtained at 4 ng ml-1 of IL-1, and the effect was smaller at 20 ng ml-1 (Wang et al. 1997). Essentially similar responses to IL-1 (1 ng ml-1) were observed in singly isolated parietal cells (inhibition by 33.8 ± 0.8 %, n = 5), suggesting that the IL-1-induced inhibition of the current (Fig. 1) does not reflect partial uncoupling of cells in isolated gastric glands. Figure 2 shows that the current- membrane potential (I-Vm) relationship of the IL-1 (1 ng ml-1)-decreased Cl- current was non-rectifying, as we have previously shown for the whole-cell Cl- current inhibited by intracellular addition of GTP
S (Sakai & Takeguchi, 1993).
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Figure 1. Inhibition of the whole-cell Cl- current of rabbit gastric parietal cells by IL-1 A, typical traces of the whole-cell Cl- current measured with the 133K+-13Cl- pipette solution and the 133K+-142Cl- bathing solution at the holding potential of 0 mV. The bathing solutions supplemented with 0.3-10 ng ml-1 of IL-1 | ||
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Figure 2. Relations between the whole-cell Cl- current (I) and membrane potential (Vm) A, typical current traces measured with the 140choline+-146Cl- pipette solution and the 140 choline+-146Cl- bathing solution. Vm was changed by a step of ±20 mV from the holding potential of 0 mV. Whole-cell Cl- currents were recorded just before (control; left) and 10 min after the addition of 1 ng ml-1 IL-1 | ||
Noise analysis of the whole-cell Cl- current
Single Cl- channel events could not be observed in 200 cell-attached and 100 excised patches of the basolateral membrane of rabbit gastric parietal cells as reported previously (Sakai et al. 1989, 1992). Therefore, the unit conductance of the Cl- channel was calculated from variances of the current fluctuation during the period of IL-1 (1 ng ml-1)-induced inhibition process (Fig. 3A). For these experiments, the zero-current potential was -30 mV under the experimental conditions and the clamped voltage was 0 mV. Thus, the intercept of the plot in Fig. 3B with the y-axis gives a unit amplitude of the single Cl- channel current corresponding to a unit conductance of 0.31 pS. Mean values with S.E.M. of the single channel conductance and total number of the channels (N) were 0.37 ± 0.02 pS and (3.58 ± 1.16) 104, respectively (n = 4). These values are not significantly different (P > 0.05) from the previous values calculated from the GTPS-elicited inhibition process of the Cl- current (0.37 ± 0.05 pS and (2.52 ± 0.44) 104; Sakai & Takeguchi, 1993, 1994).
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Figure 3. Variance noise analysis of the whole-cell Cl- current A, inhibition of the whole-cell Cl- current by 1 ng ml-1 IL-1 | ||
Specificity of action of IL-1 on the gastric parietal cell
The presence of anti-IL-1 antibody (2 µg ml-1) abolished the inhibitory effect of IL-1 (1 ng ml-1) (Fig. 4B and D), while that of control goat IgG (2 µg ml-1) did not significantly affect the IL-1-induced effect (Fig. 4C and D). The recombinant IL-1 receptor antagonist (IL-1ra; 500 ng ml-1) also abolished the effect of IL-1 (Fig. 4E and F). These results suggest that IL-1 specifically acts via a receptor in the basolateral membrane of the parietal cell. Also, corresponding to these electrophysiological results, Northern blot analysis showed that IL-1 receptor mRNA is highly expressed in rabbit gastric parietal cells (Fig. 5). The size of rabbit IL-1 receptor mRNA (5.1 kb) is comparable to human (5.1 kb) and rat (5.7 kb) IL-1 receptor mRNAs (Piquette et al. 1994; Schepp et al. 1998).
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Figure 4. Effects of anti-IL-1 A-C, IL-1 | ||
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Figure 5. Northern blot analysis for detecting rabbit IL-1 receptor mRNA Detection of rabbit IL-1 receptor (type I) mRNA on poly A+ RNAs (2.5 µg) of purified gastric parietal cells (left lane) and isolated gastric mucosa (right lane). A single band of 5.1 kb was detected (upper panel). The membrane was reprobed with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe as an RNA loading control (1.3 kb; lower panel). | ||
Involvement of the G protein-coupled production of superoxide anion (O2-) in the IL-1-elicited process
In rabbit gastric parietal cells, we have previously shown that a G protein inhibits the small-conductance Cl- channel via intracellular production of O2- (Sakai & Takeguchi, 1993, 1994, 1995). Here, we tested if this pathway is elicited by addition of IL-1. Interestingly, intracellular addition of GDPS (500 µM) abolished the IL-1 (1 ng ml-1)-induced inhibition of the Cl- current (Fig. 6). Messenger RNAs for pertussis toxin-insensitive heterotrimeric G proteins G
12 and G13 were expressed in rabbit gastric parietal cells (data not shown). Figure 7 shows that intracellular addition of superoxide dismutase (100 units ml-1), a scavenger of O2-, abolished the IL-1 (1 ng ml-1)-induced effect, while catalase (100 units ml-1), which is a scavenger of hydrogen peroxide, did not abolish it.
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Figure 6. Effect of GDP A and B, typical traces of the whole-cell Cl- current recorded at 0 mV. The 133K+-13Cl- pipette solution was supplemented without (A) or with (B) 500 µM GDP | ||
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Figure 7. Effects of superoxide dismutase (SOD) and catalase on the IL-1 A-C, typical traces of the whole-cell Cl- current recorded at 0 mV. The 133K+-13Cl- pipette solution was supplemented without (A) or with 100 units ml-1 SOD (B) or 100 units ml-1 catalase (C). The 133K+-142Cl- bathing solution supplemented with 1 ng ml-1 IL-1 | ||
The following results suggest that the G protein Gq, which activates Ca2+ release and mobilisation of protein kinase C in these cells, is not involved in this response. In the Fura-2-loaded single parietal cells, IL-1 (1 ng ml-1) did not increase [Ca2+]i (Fig. 8), whereas carbachol (50 µM) induced significant increase in [Ca2+]i (Fig. 8) as reported previously (Negulescu & Machen, 1988). Treatment of parietal cells with staurosporine (100 nM), an inhibitor of protein kinase C, for 80-100 min did not significantly affect the IL-1 (1 ng ml-1)-induced inhibition of the whole-cell Cl- current: that is, when assessed 10 min after the addition of IL-1, the inhibition percentage was 42.8 ± 9.5 % in the staurosporine-untreated cells and 38.6 ± 4.8 % in the staurosporine-treated cells (n = 3, P > 0.05).
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Figure 8. Effects of IL-1 A, a typical trace of change in [Ca2+]i of the Fura-2-loaded single parietal cells in isolated gastric glands. The bathing solutions supplemented with 1 ng ml-1 IL-1 | ||
Measurement of intracellular production of oxygen radicals
Does IL-1 really stimulate the production of O2-? To visualise this effect directly, we prepared the isolated rabbit gastric glands loaded with the oxidant-sensing fluorescent probe, H2FDA. It has been reported that intracellular O2- formation could oxidise H2FDA (Hempel et al. 1999). In the H2FDA-loaded single parietal cells, IL-1 (0.3-10 ng ml-1) increased the production of oxygen radicals (Fig. 9). Maximal effect was observed at 1-3 ng ml-1 of IL-1 (Fig. 9B). In accordance with the results of whole-cell Cl- current (Fig. 4), the IL-1-stimulated production of oxygen radicals was significantly inhibited by the presence of anti-IL-1 antibody (Fig. 10A-C) or recombinant IL-1 receptor antagonist (IL-1ra; Fig. 10D-F).
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Figure 9. Effects of IL-1 A, typical traces of increases in the fluorescein fluorescence of the H2FDA-loaded single parietal cells in isolated gastric glands. The bathing solutions supplemented with 0.3-10 ng ml-1 of IL-1 | ||
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Figure 10. Effects of anti-IL-1 A and B, typical traces of increases in the fluorescein fluorescence of the H2FDA-loaded single parietal cells in isolated gastric glands. IL-1 | ||
Rho/Rho-kinase-dependent production of O2-
Figure 11 shows that treatment of the parietal cells with Y-27632 (0.3-10 µM), a specific inhibitor of Rho-kinase, for 80-100 min concentration-dependently attenuated the IL-1 (1 ng ml-1)-induced inhibition of the Cl- current. Treatment of the cells with Y-27632 (0.3-10 µM) for 70-100 min also inhibited the IL-1-stimulated production of oxygen radicals in the parietal cell (Fig. 12). In both cases, significant effects of Y-27632 were observed at 1-10 µM. Intracellular addition of C3 exoenzyme (1 µg ml-1), which ADP-ribosylates and inactivates Rho (Wilde & Aktories, 2001), abolished the IL-1 (1 ng ml-1)-induced inhibition of the whole-cell Cl- current: that is, when assessed 10 min after the addition of IL-1, the inhibition percentages in the presence and absence of C3 exoenzyme were 1.7 ± 1.6 and 36.2 ± 4.6 %, respectively (n = 4, P < 0.01). Statins (HMG-CoA reductase inhibitors) have been reported to indirectly inhibit Rho by preventing its essential geranylation (Hausding et al. 2000). Figure 13 shows that fluvastatin (10 µM), a lipophilic statin, significantly inhibited the IL-1-induced effects on the channel activity and production of oxygen radicals.
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Figure 11. Effect of Y-27632 on the IL-1 A and B, typical traces of the whole-cell Cl- current recorded at 0 mV. Cells were pre-incubated without (A) or with (B) 10 µM Y-27632 for 80-100 min at 35 °C. The 133K+-142Cl- bathing solutions supplemented with 1 ng ml-1 IL-1 | ||
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Figure 12. Inhibitory effect of Y-27632 on the IL-1 A and B, typical traces of increases in the fluorescein fluorescence of the H2FDA-loaded single parietal cells. Cells were pre-incubated without (A) or with (B) 10 µM Y-27632 for 70-100 min at 35 °C. The bathing solutions supplemented with 1 ng ml-1 IL-1 | ||
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Figure 13. Inhibition of the IL-1-elicited effects by fluvastatin A and B, typical traces of the whole-cell Cl- current recorded at 0 mV. Cells were pre-incubated without (A) or with (B) 10 µM fluvastatin for 60-90 min at 35 °C. The 133K+-142Cl- bathing solutions supplemented with 1 ng ml-1 IL-1 | ||
Activation of Rho by IL-1
Here we examined whether IL-1 activates Rho in rabbit gastric parietal cells using an assay based on selective precipitation of activated (i.e. GTP-bound) Rho. Figure 14 shows that IL-1 concentration-dependently activated Rho. It should be noted that maximal activation of Rho was observed at 1-3 ng ml-1 of IL-1 (Fig. 14), in accordance with the results of whole-cell Cl- current (Fig. 1) and production of oxygen radicals (Fig. 9).
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Figure 14. Activation of Rho protein by IL-1 Rabbit parietal cells (2 | ||
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DISCUSSION |
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In the present study using rabbit gastric parietal cells, we have shown that IL-1 acts as a ligand for inhibition of the basolateral small-conductance Cl- channel via the G protein-mediated production of O2-: that is, (1) electrophysiological and pharmacological properties of the IL-1-sensitive whole-cell Cl- current (Figs 2, 3, 6 and 7) are consistent with those of the GTPS-sensitive Cl- current as previously reported (Sakai & Takeguchi, 1993, 1994); (2) IL-1 stimulated the intracellular production of oxygen radicals in the H2FDA-loaded parietal cells (Fig. 9); (3) the IL-1-induced maximal effects for both the whole-cell Cl- current and the production of oxygen radicals were observed at the same concentration (1-3 ng ml-1; Fig. 1 and Fig. 9); (4) IL-1-induced effects were abolished by anti-IL-1 antibody and recombinant IL-1ra (Fig. 4 and Fig. 10); and (5) IL-1 receptor mRNA was highly expressed in rabbit gastric parietal cells (Fig. 5).
It has been suggested that IL-1 induced by Helicobacter pylori infection is a mediator of gastric acid inhibition (Takeshima et al. 2001). IL-1 directly inhibited the carbachol-stimulated acid secretion in isolated gastric parietal cells of rat (Schepp et al. 1998) and rabbit (Beales & Calam, 1998, 2001). This inhibitory action was pertussis toxin insensitive (Beales & Calam, 1998) and completely reversed by several inhibitors of protein kinase C (Beales & Calam, 2001). The action of IL-1 was independent of protein kinase A and protein kinase G activity (Beales & Calam, 2001). In the present study, we found that inhibition of the basolateral small-conductance Cl- channel in rabbit gastric parietal cells is not mediated by protein kinase C (see Results concerning staurosporine), but mediated by Rho/Rho-dependent kinase (Figs 11-14). Taken together, these results suggest that the IL-1-elicited pathway for inhibition of the Cl- channel is different from that for gastric acid secretion.
How does the Rho/Rho-kinase stimulate O2- production in rabbit parietal cells? In human polymorphonuclear leukocytes, the Rho-kinase-mediated production of O2- has been reported to involve NADPH oxidase (Kawaguchi et al. 2000). However, it is unlikely that NADPH oxidase in the plasma membrane contributes to the present mechanism, because it produces O2- at the extracellular side. Possible candidates include the cytochrome P450 (CYP) enzymes of the microsome membranes. The CYP reaction cycle through unproductive pathways can give rise to side products such as O2-, H2O2 and water (Puntarulo & Cederbaum, 1998). In fact, the production of O2- by human CYP enzymes was directly measured (Puntarulo & Cederbaum, 1998). Although CYP enzymes are most abundant in the liver, it has been reported that many kinds of the enzymes are expressed in extrahepatic tissues including gastrointestinal tracts (Ding & Kaminsky, 2003). Recent studies have demonstrated the expression of CYP2C (Yokose et al. 1999), CYP2J (Zeldin et al. 1997) and CYP2S1 (Rylander et al. 2001) in human stomach mucosa. Apparently, further studies are necessary to show direct connection between O2- production and CYP enzymes in the present system.
So far, a variety of interleukins have been reported to regulate the activity of epithelial Cl- channels. IL-5 inhibited cAMP-dependent Cl- currents in the human biliary cell line Mz-ChA-1 (McGill et al. 2001). IL-2 increased cAMP-dependent Cl- secretion in cultured human small intestinal enterocytes (O'Loughlin et al. 2001). IL-4 diminished cAMP- and Ca2+-mediated Cl- secretion in the human intestinal cell line T84 (Zünd et al. 1996). Among these reports, the effects of IL-2 and IL-4 were mediated by change in transcription, which generally requires several hours of treatment (Zünd et al. 1996; O'Loughlin et al. 2001), whereas the effect of IL-5 was observed immediately (McGill et al. 2001). In the present study, the basolateral Cl- channel in rabbit gastric parietal cells was found to be regulated immediately by IL-1. It should be noted that neither cAMP nor Ca2+ (Sakai & Takeguchi, 1993) mediate inhibition of this Cl- channel.
We found previously that prostaglandin E2 binds to the EP3 receptor in the basolateral membrane of the rabbit gastric parietal cell, resulting in an increase in [Ca2+]i, which sequentially activates constitutive NO synthase and guanylate cyclase and increases the intracellular cGMP level. This increased level of cGMP in turn activates the parietal cell basolateral small-conductance Cl- channel in the parietal cells (Sakai et al. 1995, 1996). A similar Cl- channel has also been found in rat gastric parietal cells (Ikari et al. 1999). Prostaglandin E2 was shown to have cytoprotective effects against ethanol in isolated rabbit gastric parietal cells (Barr et al. 1988). We have recently found that opening of the basolateral Cl- channel via the prostaglandin E2-elicited NO/cGMP pathway is closely associated with cytoprotection against ethanol-induced cytotoxicity in the parietal cells (Sakai et al. 1998). On the other hand, we have found in the present study that IL-1 inhibits the basolateral small-conductance Cl- channel via intracellular production of O2-. Our previous study showed that very short treatment of the parietal cells (for only 30 s) with NPPB, a Cl- channel blocker, had a cytotoxic effect, which may be related to a closure of the basolateral Cl- channel (Sakai et al. 1998).
The proinflammatory cytokine IL-1 is emerging as a key mediator of the Helicobacter pylori-associated diseases in stomach (El-Omar, 2001), and both IL-1 and gastric acid are essential to cause recurrence of gastric ulcers (Watanabe et al. 2001). IL-1 mRNA was not detected in the normal or intact mucosa of ulcerated stomachs, but its expression was induced in the ulcerated tissue (Takahashi et al. 1999). In contrast, IL-1 has been reported to reduce gastric injury by ethanol, and this effect was mediated by stimulated production of prostaglandin E2 in the stomach (Robert et al. 1991). Such diverse actions of IL-1 suggest that IL-1 contributes to both the pathogenesis of inflammation and the healing process which restores the integrity of the gastric mucosa (Ernst et al. 1997; El-Omar, 2001). These two distinct actions of IL-1 may be paralleled with the activity of the basolateral Cl- channel of the parietal cells: that is, IL-1 acts directly on the parietal cell and inhibits the Cl- channel at the inflammatory stage, while IL-1-released prostaglandin E2 activates the Cl- channel at the healing stage.
In summary, in the present study we have found that proinflammatory IL-1 binds to the IL-1 receptor in the basolateral membrane of rabbit gastric parietal cells, and inhibits the basolateral small-conductance Cl- channel via the G protein-mediated Rho/Rho-kinase-dependent production of O2-. The negative regulation of this Cl- channel may be associated with inflammatory diseases in the stomach in contrast to positive regulation of the channel which contributes to the cytoprotective mechanism.
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Acknowledgements
We thank Dr Andrew F. James (University of Bristol, Bristol, UK) for his careful reading of this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (to H.S. and N.T.) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.S. and N.T.), and by the grants from Uehara Memorial Foundation, Takeda Science Foundation, Suzuken Memorial Foundation, The Research Foundation for Pharmaceutical Sciences and Tamura Foundation for Promotion of Science and Technology (to H.S.). T. Suzuki is a research fellow of Japan Society for the Promotion of Science.
H. Sakai, Y. Ohira, A. Tanaka and T. Suzuki contributed equally to this work
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) and 10 min after the addition of 1 ng ml-1 IL-1
). * Significantly different from the value in the absence of IL-1
[Ca2+]i (= peak value - basal value) from 5 experiments. NS, not significantly different (P > 0.05) from the level before the addition of IL-1