1Division of Gastroenterology, Department of Medicine, University of California San Diego, San Diego, California 92103; and 2Division of Digestive Diseases, University of Cincinnati and Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0595
Submitted 21 February 2003 ; accepted in final form 17 July 2003
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ABSTRACT |
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acid-stimulated bicarbonate secretion; extracellular signal-related kinase phosphorylation; intracellular signaling
Given that uroguanylin and guanylin are potent stimuli for duodenal secretion (13, 21), we postulated that GC-C likely played a key role in the physiological stimulation of duodenal
secretion in response to luminal acidification. In addition, we hypothesized that alterations in intracellular signaling events, specifically MAP kinases, which have been shown to play a role in the regulation of Cl- secretion (22), might also be involved in the regulation of
secretion in the duodenum. To evaluate the role of transmembrane GC-C in acid-stimulated
secretion, we used a well-validated in vivo luminal perfusion method in the murine GC-C KO model. In separate experiments, the effect of acid stimulation on cGMP levels and ERK phosphorylation was examined.
The results indicate that GC-C plays an important role in secretion in the murine duodenum. The absence of transmembrane GC-C, however, did not affect the acid-mediated rise in cGMP level, suggesting that GC-C-independent pathways for cGMP generation occur with luminal acidification. Luminal acidification resulted in a rapid stimulation of ERK phosphorylation. Additionally, in contrast to the WT mice, a marked attenuation in ERK phosphorylation was seen in the GC-C KO mice. Inhibition of ERK phosphorylation in the WT mice resulted in an impaired
secretory response to luminal acidification, confirming the functional relevance of the biochemical observations.
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MATERIALS AND METHODS |
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Basal and HCl-stimulated secretion in vivo. Animals (n
6 pairs) were maintained with free access to food and water for up to 1 h before the experiment, when food was removed. Anesthesia was induced by intraperitoneal administration of 10 µl/g body wt of hypnorm/midazolam cocktail as described previously (15). The abdomen was opened by two small lateral incisions, and the proximal 47 mm of duodenum, from the pylorus to just cephalad to the entry of the common bile duct, was isolated so as not to compromise vascular supply. A small polyethylene tube (PE-50) with a distal flange was advanced to the duodenal bulb via the stomach, and a ligature was secured around the pylorus. Distal to the pylorus and just proximal to the pancreaticobiliary duct, a small incision was made and PE-50 flanged tubing was secured by another ligature to allow for drainage. The isolated duodenal segment was gently flushed and then perfused (Harvard Infusion Pump, Harvard Apparatus, South Natick, MA) at a rate of 0.17 ml/min with 154 mM NaCl (37°C). Effluents from the isolated segment were visually free of bile and blood throughout all experiments. Animals were fitted with a miniature O2 mask, maintained at 37°C, and hydrated with normal saline (50100 µl/h sc). Anesthesia was maintained using the hypnorm/midazolam cocktail at 20% of the initial dose every 3045 min as indicated by respiratory rate and toe-pinch reflex. Respiration rates were determined every 10 min, and blood was taken at the end of the study to determine plasma
. Animals could be sustained for >3 h under these experimental conditions, substantiating our earlier studies (15).
After an initial 20-min washout and recovery period, basal secretion was measured for 30 min. To examine the effect of luminal acidification, the duodenal segment was acidified with a 5-min perfusion of 10 mM HCl (made isosmolar with NaCl). The segment was then gently flushed to remove residual acid, and
secretion was measured for an additional 60 min.
could not be measured during the 5-min acid perfusion or the following 5-min period, because back-titration cannot be performed in acidified samples. Therefore, we used the pH/pCO2 method in the last 5-min period to calculate
concentration for the 15-min period. The low concentration of HCl used in these studies has previously been shown not to induce epithelial damage (15). The amount of
in the effluents was quantitated by a validated micro-back-titration method (Radiometer Analytical). Briefly, 100 µl of 50 mM HCl were added to the sample with 2 ml of distilled H2O. Samples were then gassed with N2, prewashed in Ba(OH)2, and back-titrated with 2.5 mM NaOH to an end point of pH 7.0.
outputs were determined in 15-min periods and expressed as micromoles per centimeter per hour. Stimulated
outputs are shown over a progressive time course or as net secretion calculated by subtracting the average basal
secretion from the acid-stimulated
secretion at each time point after luminal acidification. Results are expressed as means ± SE. After each experiment, the length of the duodenal test segment was measured in situ to the nearest 0.5 mm. There were no significant differences in segment lengths between the KO and WT mice.
Effect of PD-98059 on secretion in vivo. Pilot experiments revealed a decrease in levels of all tyrosine phosphorylated proteins and, specifically, phosphorylated ERK1/2 in the duodenum from the KO animals following luminal acidification. Therefore, in some experiments, the duodenum from WT and KO mice (n = 3 of each) was perfused with a selective inhibitor of ERK phosphorylation, PD-98059, at 2 x 10-5 M (Calbiochem, San Diego, CA) in the experiment described above. The dose of PD-98059 used was determined from previous studies on chloride transport in T84 cells (22). Luminal acidification and saline perfusion in the presence of PD-98059 in the perfusate followed PD-98059 pretreatment in these experiments.
Analysis of ERK phosphorylation by Western blotting. To demonstrate whether luminal acidification causes ERK phosphorylation, duodenal mucosae from WT and GC-C KO mice were exposed to HCl in vitro and analyzed by Western blotting. Initial dose-response studies (10 µM10 mM) showed that increased ERK phosphorylation was no longer seen at concentrations >1 mM HCl, and so a dose of 100 µM was used. Paired experiments were performed (n = 6). Proximal duodenum of each mouse was excised and divided into three segments. The tissues were allowed to stabilize for 1 h at 37°C in normal saline containing 10 µM indomethacin (prostaglandin synthesis inhibitor) and then treated as follows: HCl at a final concentration of 100 µM (pH 4.0) was added to all tissues except the control segment, which was used to determine basal levels of ERK phosphorylation. Tissue samples were incubated with acid for 5 min at 37°C, after which they were transferred to separate tubes containing normal saline with indomethacin and incubated for 5 or 15 min. Control segments were not exposed to acid. In some experiments (n = 3 pairs), tissues were incubated with PD-98059 for 30 min before exposure to HCl to determine the effect of the inhibitor. At each time point, tissues were transferred to tubes containing ice-cold lysis buffer (1% Triton X-100, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml antipain, 25 µl protease inhibitor cocktail, and 100 µg/ml phenylmethylsulfonylfluoride in phosphate-buffered saline) and homogenized. The homogenate was placed on an end-over-end rotator at 4°C for 45 min and then centrifuged. The supernatant was collected, and the protein concentration of the lysates was determined (BCA Protein Assay Kit, Peirce, Rockford, IL). Lysates were mixed with an equal volume of 2x gel loading buffer (100 µM Tris·HCl, pH 7.0, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol) and boiled for 2 min before SDS gel electrophoresis. An equal amount of protein was loaded from each sample in each lane. Lysates were electrophoresed using 412% NuPage Bis-Tris gels in 2-(N-morpholino) ethane sulfonic acid SDS running buffer (Invitrogen, Carlsbad, CA). Resolved proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA), and the membranes were blocked with a 3% solution of blocking buffer (Upstate Biotechnology, Lake Placid, NY) in Tris-buffered saline with 0.1% Tween (TBST) for 1 h. This was followed by incubation with anti-phospho p44/42 MAP kinase (ERK1/2) in 2% blocking buffer (Cell Signaling Technology, Beverly, MA) for 1 h at room temperature or overnight at 4°C. After membranes were washed (6 x 10 min) in TBST, they were incubated for 1 h with horseradish peroxidase-conjugated anti rabbit IgG (Transduction Laboratories, Lexington, KY) in 1% blocking buffer. The membranes were once again washed as described earlier, and the bound antibodies were detected using an enhanced chemiluminescence detection kit (ECL Plus, Amersham-Pharmacia Biotech, Piscataway, NJ). The blots were immediately exposed to X-Omat Blue XB-1 X-ray film (New England Nuclear Life Science Products, Boston, MA) for 45 s and developed. Blots were stripped and reexposed to anti ERK-1 (K-23, Santa Cruz Biotechnology, Santa Cruz, CA) to ensure that comparable levels of ERK protein were present in all samples. Densitometric analysis of blots was carried out using the program Scion Image after scanning the X-ray films using the DeskScanII scanning program.
Measurement of cGMP accumulation in duodenal mucosa in vitro. The proximal 1 cm of mouse duodenum was excised, divided into two segments longitudinally, and each was placed in HBSS (GIBCOBRL, Grand Island, NY) containing 0.1 mM of the phosphodiesterase inhibitor IBMX (Calbiochem) for 10 min at 37°C. One segment was treated with HCl (10 mM) for 5 min, whereas the control segment remained in HBSS/IBMX. Each tissue was then transferred to fresh HBSS/IBMX for an additional 5 min. The tissue and final two HBSS washes were combined and homogenized (acid-containing samples were first neutralized with sodium hydroxide). Aliquots of the homogenates were assayed for protein by the bicinchoninic acid method (Pierce, Rockford, IL). cGMP was extracted using Amprep SAX anion-exchange columns (Amersham Pharmacia Biotech). Samples were acetylated and quantified using Biotrak cGMP EIA (Amersham Pharmacia Biotech).
Statistical analyses. All data are expressed as means ± SE for a series of n experiments. Student's t-tests, ANOVA with the Student-Newman-Keul's test, or the nonparametric Dunn's multiple comparison test was used to compare mean values as appropriate. P values <0.05 were considered to represent statistical significance.
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RESULTS |
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secretion in WT and GC-C KO mice in vivo. Basal
secretion was significantly diminished in GC-C KO mice compared with WT animals (2.6 ± 0.5 vs. 5.0 ± 0.9 µmol/·cm-1·h-1, respectively, n
6, P < 0.05). Peak
secretion in response to HCl occurred within 15 min in both WT and KO mice. However, the peak response in KO mice was significantly impaired compared with WT mice (7.2 ± 1.1 vs. 16.4 ± 2.4 µmol/·cm-1·h-1, respectively, P < 0.001; Fig. 1). In addition, whereas
secretion returned to basal levels in WT mice by 45 min, levels in KO mice remained elevated to the end of the time course at 60 min. These data suggest that the GC-C receptor is involved in basal as well as acid-stimulated
secretion.
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Effect of PD-98059 on secretory response of WT mice. To determine whether ERK phosphorylation is required for
secretion, PD-98059, a selective inhibitor of MEK 1/2, the kinases that activate ERK via phosphorylation, was used. The duodena of WT and GC-C KO mice were perfused in vivo with PD-98059 before luminal acidification. PD-98059 did not alter basal
secretion (WT with PD-98059 vs. without, 4.2 ± 0.4 vs. 5.0 ± 0.9; KO with PD-98059 vs. without, 4.4 ± 1.2 vs. 2.6 ± 0.5). However, the net acid-stimulated
secretory response in WT mice was significantly inhibited by the presence of PD-98059 (Fig. 2). In contrast, PD-98059 did not significantly alter the response to HCl in the GC-C KO mice. These results suggest that acid-stimulated
secretion is partially dependent on phosphorylation of ERK in WT mice. To further substantiate these results, we next examined whether ERK phosphorylation occurs in acid-stimulated tissue in vitro.
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Effect of acid stimulation on ERK phosphorylation in vitro. To determine whether acidification stimulates ERK phosphorylation, duodenal mucosae from WT and GC-C KO mice were exposed to acid in vitro and the time course of ERK phosphorylation was analyzed by Western blotting (Fig. 3). In pilot experiments, an attenuation in levels of all tyrosine-phosphorylated proteins and specifically phosphorylated ERK was seen in the duodenum from KO animals following in vivo exposure to HCl (data not shown). In subsequent in vitro experiments, a rapid and time-dependent phosphorylation of ERK on acidifi-cation was seen in the duodena of WT mice. A significant increase in ERK phosphorylation was seen 5 min following acid exposure, which then decreased by 15 min. In contrast, no increase over basal levels was apparent in HCl-stimulated tissue from GC-C KO mice. Comparable levels of ERK protein were present in all the samples (n = 6, Fig. 3A).
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Densitometric analysis of the Western blots indicated that ERK phosphorylation was stimulated in a statistically significant fashion by acid exposure in the WT but not KO mouse tissues (Fig. 3B), suggesting that defective phosphorylation of ERK may contribute to the impaired acid-stimulated secretion in the KO animals. Furthermore, both basal and acid-evoked ERK phosphorylation was abolished by PD-98059 in duodenal mucosae from both WT and KO mice (Fig. 4).
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Analysis of cGMP levels in duodena of WT and GC-C KO mice. We next examined whether the attenuation in secretion and the decrease in levels of phosphorylated ERK in the KO animals were accompanied by decreased cGMP levels due to the absence of GC-C. Generation of cGMP was measured in response to HCl exposure in duodenal mucosae from WT and KO animals in vitro. Surprisingly, basal levels of cGMP extracted from homogenized whole thickness duodenal segments were similar in WT and KO mice. Moreover, a statistically significant increase in cGMP, comparable in magnitude, occurred in response to a 5-min HCl exposure in both WT and KO animals (Table 2).
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DISCUSSION |
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The intestinal receptor for E. coli heat-stable toxin STa is a plasma membrane isoform of guanylate cyclase, GC-C (12, 31). In a form of unique molecular mimicry, binding of bacterial STa peptide to this mammalian receptor has been shown to increase fluid and electrolyte secretion in the small and large intestine, the pathogenic mechanism for E. coli-induced traveler's diarrhea. Transmembrane GC-C is primarily expressed in both crypt and villus enterocytes of the gastrointestinal tract of man and mouse (23, 37). Guanylin and uroguanylin, the endogenous ligands for GC-C, are also expressed in high levels in the intestine. At the mRNA level, in the mouse and rat intestine, uroguanylin is more highly expressed in the proximal gut, whereas guanylin is found in higher levels in the distal gut (7). Hence, several studies have postulated a key role for GC-C in regulating fluid and electrolyte homeostasis in the intestine (8). In particular, GC-C activation by uroguanylin has been identified to stimulate transport in the mouse duodenum (21). Recently, it has been shown that GC-C directly associates with a postsynaptic density protein 95/drosophila disks large/zona occludens-1 (PDZ) domain-containing protein at the apical surface of intestinal epithelial cells (32). Thus GC-C may participate in a complex consisting of several PDZ binding proteins including CFTR (34) that serves to compartmentalize signaling molecules and effector proteins.
Studies in animal models and T84 cells have revealed that STa, guanylin, and uroguanylin stimulate Cl- secretion by increasing intracellular cGMP (9, 10, 19). Events downstream of cGMP release have been studied using cGMP kinase II KO mice. In these animals, resistance to the effects of cholera toxin was seen in Ussing-chamber studies of the small intestine, suggesting that Cl- secretion in the murine jejunum involves activation of cGMP kinase II, which, in turn, is believed to mediate electrolyte movement through the final common pathway of the CFTR (25, 28, 33, 39). Although direct evidence is lacking, secretion stimulated by STa and uroguanylin is believed to occur via similar pathways, although regulation of other transporters involved in
secretion, such as the apical Cl-/
exchanger (20), is also possible.
Disruption by homologous recombination of the gene encoding the GC-C led to the development of a mouse model in which there is complete absence of the GC-C mRNA and its protein product (24). In this study, we have used this model to define the physiological role of GC-C signaling pathways in duodenal secretion. The work presented here suggests that acid-stimulated
secretion in the murine duodenum in vivo closely involves the GC-C receptor. Although the
secretory response to acid was not completely abolished, the significant attenuation seen in the GC-C KO animals suggests a major role for signaling via transmembrane GC-C in this process. Acid-stimulated
secretion is believed to occur as a consequence of multiple pathways, including the luminal release of PGE2 as well as stimulation of enteric nerves with the release of VIP, nitric oxide (NO), and other as yet unidentified mediators (13). Most likely a redundancy of pathways exists, as is found in other physiological events, which would explain the attenuation rather than abolishment of acid-stimulated
secretion in the GC-C KO animals.
A significant elevation in cGMP with acid exposure was seen in the KO animals, comparable with that seen in the WT mice despite the absence of transmembrane GC-C in the former. There is intestinal expression of other members of the guanylyl cyclase family, including GC-A, a receptor for atrial natriuretic peptide (38), and soluble guanylyl cyclase, which is activated by intracellular molecules such as NO and hydroxyl radicals (6). Recent studies have demonstrated the involvement of the L-arginine/NO axis in acid-stimulated secretion as well (16, 17).
Hence, in our studies, the significant elevation of cGMP seen in the KO animals may indeed be related to the existence of other forms of GC in the duodenal mucosa. The in vitro experiments performed to assess HCl-stimulated cGMP release were performed on unstripped duodenal tissue, and they inherently lack the specificity of the in vivo experiments where mucosal acidification was achieved. The cGMP release seen in the KO animal mucosae in vitro could therefore be from nonepithelial components of the tissue, such as smooth muscle sites. Therefore, whereas it is attractive to hypothesize noncGMP-mediated mechanisms being involved in the GC-C signaling of secretion, on the basis of our data, this would be extremely speculative. A second signaling pathway has been suggested by reports that STa stimulates inositol phospholipid breakdown and that the resulting rise in inositol triphosphate precedes a rise in intracellular calcium in rat enterocytes (5, 18). Alternatively, as GC-C may participate in a signaling complex via its interactions with PDZ domain scaffold proteins, a compartmentalized loss of cGMP signaling, not discernible by our determination of total tissue cGMP levels, could contribute to the decrease in
secretion.
Intracellular signal-transduction events mediating secretion are poorly understood, because work thus far has concentrated on physiological study of animal or human models. There is, however, precedence for the involvement of intracellular ERK in ion transporter regulation. ERK activation has been demonstrated to regulate Na+/H+ exchange, either positively, as in the rat myocardium (27), or negatively, in the case of mediating nerve growth factor inhibition of Na+/H+ exchanger (NHE1) in the medullary thick ascending limb of the kidney (41). In the renal proximal tubule, ERK is known to mediate angiotensin II stimulation of NBC1, the basolateral Na-
cotransporter (30). In the T84 colonic epithelial cell line, transactivation of the epidermal growth factor receptor results in ERK activation and inhibition of calcium-mediated chloride secretion (22). Other studies by Soh et al. (35, 36) in colon tumor cells have demonstrated that cGMP signal transduction involves the MEKK1/JNK pathway. Similar to these examples, our data suggest that the GC-C receptor may function as a signaling molecule to mediate HCl-stimulated
secretion via ERK phosphorylation. Future experiments including the study of growth factor-induced ERK phosphorylation and the identification of the transporter proteins regulated by ERK activation in this system will allow a better understanding of the duodenal response to acidification.
Our data establish a potential role for the ERK1/2 isoforms of MAP kinase in epithelial secretion. Treatment with PD-98059, a specific inhibitor of ERK phosphorylation, resulted in inhibition of acid-stimulated
secretion in WT mice, suggesting that activation of ERK is causal in the acid-stimulated
secretory response. More importantly, our data implicate transmembrane GC-C expression as a necessary upstream facilitator of ERK activation. The regulatory event that is altered in this model is phosphorylation, because normal levels of ERK protein are present in the duodenum of KO animals. The presence of indomethacin in the incubation and subsequent solutions for these studies rules out the possibility of endogenous prostaglandin release contributing to the acid stimulation of ERK phosphorylation. Mechanisms by which GC-C may affect ERK1/2 activation, as well as the events downstream of ERK phosphorylation that lead to stimulation of
secretion, remain to be studied.
In summary, transmembrane GC-C expression is of key importance in the normal duodenal secretory response to acid-stimulation in vivo. Although events downstream of GC-C that mediate the acid-stimulated response in secretion remain to be fully defined, they appear to involve activation of the ERK1/2 isoforms of MAP kinase.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (5RO1DK-33491 to JII; 5KO8DK-02517 to V. S. Pratha) and a Veteran Affairs grant (5393108 to R. A. Giannella).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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