A role for guanylate cyclase C in acid-stimulated duodenal mucosal bicarbonate secretion

S. P. Rao,1 Z. Sellers,1 D. L. Crombie,1 D. L. Hogan,1 E. A. Mann,2 D. Childs,1 S. Keely,1 M. Sheil-Puopolo,2 R. A. Giannella,2 K. E. Barrett,1 J. I. Isenberg,1 and V. S. Pratha

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luminal acidification provides the strongest physiological stimulus for duodenal secretion. Various neurohumoral mechanisms are believed to play a role in acid-stimulated secretion. Previous studies in the rat and human duodenum have shown that guanylin and Escherichia coli heat-stable toxin, both ligands of the transmembrane guanylyl cyclase receptor [guanylate cyclase C (GC-C)], are potent stimulators for duodenal secretion. We postulated that the GC-C receptor plays an important role in acid-stimulated secretion. In vivo perfusion studies performed in wild-type (WT) and GC-C knockout (KO) mice indicated that acid-stimulated duodenal secretion was significantly decreased in the GC-C KO animals compared with the WT counterparts. Pretreatment with PD-98059, an MEK inhibitor, resulted in attenuation of duodenal secretion in response to acid stimulation in the WT mice with no further effect in the KO mice. In vitro cGMP generation studies demonstrated a significant and comparable increase in cGMP levels on acid exposure in the duodenum of both WT and KO mice. In addition, a rapid, time-dependent phosphorylation of ERK was observed with acid exposure in the duodenum of WT mice, whereas a marked attenuation in ERK phosphorylation was observed in the KO animals despite equivalent levels of ERK in both groups of animals. On the basis of these studies, we conclude that transmembrane GC-C is a key mediator of acid-stimulated duodenal secretion. Furthermore, ERK phosphorylation may be an important intracellular mediator of duodenal secretion.

acid-stimulated bicarbonate secretion; extracellular signal-related kinase phosphorylation; intracellular signaling


EPITHELIAL BICARBONATE () secretion contributes to the health of the duodenal mucosa in the face of extremely low luminal pH from the large quantities of hydrochloric acid (HCl) secreted by the stomach. Similar to other secretory epithelia, cAMP-, cGMP- and calcium-dependent mechanisms are believed to mediate the action of the well-described secretogogues for duodenal secretion (11, 26). Guanylin and uroguanylin, endogenous ligands for the transmembrane guanylate cyclase C receptor (GC-C), are known to be potent stimuli for duodenal secretion (13, 21). Contrary to other agents, such as PGE2, the GC-C ligands guanylin and Escherichia coli heat-stable toxin (STa) have been shown to stimulate greater than Cl- flux in the duodenum (13). GC-C is known to increase intracellular cGMP levels, which results in well-defined signal-transduction events mediated via protein kinases (39, 40). The events mediating GC-C-mediated secretion in the duodenum, although less well defined, are believed to involve cGMP generation and the cGMP-mediated stimulation of CFTR (33).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. A murine GC-C knockout (KO) model was used. As previously described (24), the mouse gene encoding GC-C was disrupted at the 5'-end. Mice heterozygous for the disrupted GC-C allele (hybrids of 129/Sv and Black Swiss) were backcrossed for five generations to the C57BL/6 inbred strain. These backcrossed heterozygous GC-C (-/+) mice were mated to produce homozygous GC-C (-/-) and GC-C (+/+) mice. The mice used in these experiments are their progeny. The absence of GC-C mRNA and protein was verified by Northern blot, RT-PCR, and Western blot analysis. Histological examination revealed that the major organ systems were normal. GC-C (-/-) mice do not exhibit STa-induced secretion in the suckling mouse assay nor is colonic electrolyte transport affected by STa (4).

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 4–7 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 (50–100 µl/h sc). Anesthesia was maintained using the hypnorm/midazolam cocktail at 20% of the initial dose every 30–45 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 µM–10 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 4–12% 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phenotypic characteristics of WT and GC-C KO mice. No major phenotypic differences were observed between the WT and GC-C KO animals. The WT and KO animals were matched for age and weight as shown in Table 1. Furthermore, there were no significant differences in the respiratory rates and end-of-study plasma concentrations between the WT and KO mice (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Summary of experimental parameters in WT and GC-C-KO animals

 

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.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Net acid-stimulated proximal duodenal mucosal secretion in wild-type (WT; n = 7) and guanylate cyclase C (GC-C) knockout (KO; n = 6) mice in vivo. After basal secretion was measured for 30 min, the duodenum was acidified with 10 mM HCl (8.5 µmol; isomolar with NaCl) for 5 min. Net secretion was calculated by subtracting the average basal value from the total value obtained at each time point. Results are expressed as means ± SE. Asterisks denote values that differ significantly between WT and KO mice (*P < 0.05; **P < 0.001).

 

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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Effect of PD-98059 on acid-stimulated secretion in WT and GC-C KO mice in vivo. WT and KO mice (n = 3 pairs) were perfused with PD-98059 (2 x 10-5 M) before luminal acidification and then analyzed for secretion as in Fig. 1. Perfusion with PD-98059 resulted in a significant decrease in net acid-stimulated secretion in WT mice compared with those that were not exposed (*P < 0.05). In contrast, in the GC-C KO mice, there was no significant alteration in net acid-stimulated secretion following PD-98059. Results are means ± SE.

 

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).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3. Western blot analysis of ERK phosphorylation in duodenal tissues from WT and GC-C KO mice in response to acidification in vitro. Tissues were exposed to saline or 100 µM HCl for 5 min. After exposure to acid, incubation in normal saline was continued for 5 or 15 min as indicated. A: representative blots from 6 similar experiments. Top shows the results from a blot probed for phosphorylated ERK. Bottom shows the same blot was exposed to anti-ERK-1 to show that comparable levels of ERK protein were present in all samples. B: densitometric analysis of 6 similar blots probed for phosphorylated ERK. The asterisk denotes a value that is significantly higher than the corresponding value in KO mice (means ± SE; *P < 0.05). B, basal; AU, arbitrary units.

 

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).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Effect of PD-98059 on acid-stimulated ERK phosphorylation in duodenal mucosae from WT (A) and GC-C KO (B) mice. Exposure of duodenal tissue segments to PD-98059 (2 x 10-5 M) for 30 min before acid exposure resulted in complete inhibition of ERK phosphorylation compared with non-treated segments (*P < 0.05). Western blots were performed as described in MATERIALS AND METHODS, and a densitometric analysis of several such blots is shown. Results are means ± SE for 3 animals in each case.

 

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).


View this table:
[in this window]
[in a new window]
 
Table 2. Generation of cGMP in duodenal tissue exposed to HCl in vitro

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Duodenal mucosal secretion (DMBS) occurs under basal and agonist-stimulated conditions. Although a wide range of agonists has been shown to stimulate active secretion, acidification of the duodenal lumen by HCl is believed to be the most relevant physiological stimulus (14). Multiple effectors are believed to mediate acid-stimulated secretion, including VIP, luminal release of PGE2, and neurogenic factors (1). Our study additionally establishes a key role for transmembrane GC-C in acid-stimulated DMBS in vivo. In mice lacking GC-C, basal DMBS was compromised and luminal acidification also resulted in a secretory response that was significantly attenuated compared with the WT controls. However, a significant and comparable increase in cGMP was seen in both the WT and KO duodenum on exposure to HCl in vitro, suggesting alternate GC-C-independent pathways for cGMP generation. In vitro studies also demonstrated a rapid, time-dependent (within 5 min) phosphorylation and activation of the ERK1/2 isoforms of MAP kinase in WT duodenal mucosa on treatment with HCl. Pretreatment with the MEK inhibitor PD-98059 was associated with attenuation of the secretory response to acid in the WT animals in vivo and a corresponding inhibition of acid-stimulated ERK phosphorylation in vitro. Thus pretreatment of WT animals with PD-98059 resulted in a response that was comparable with the blunted acid-stimulated response seen in the KO animals. These findings suggest that, similar to Cl- secretion (22), ERK activation plays an important role in the regulation of duodenal secretion.

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.


    ACKNOWLEDGMENTS
 
We thank Dr. J. Steinbach for assistance with statistical analysis.

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).


    FOOTNOTES
 

Address for reprint requests and other correspondence: Vijayalakshmi S. V. Pratha, Division of Gastroenterology (8413), UCSD Medical Center, 200 West Arbor Dr., San Diego, CA 92103–8413 (E-mail: vpratha{at}calincresearch.com).

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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Allen A, Flemstreom G, Garner A, and Kivilaakso E. Gastroduodenal mucosal protection. Physiol Rev 73: 823-857, 1993.[Abstract/Free Full Text]
  2. Ballesteros MA, Wolosin JD, Hogan DL, Koss MA, and Isenberg JI. Cholinergic regulation of human proximal duodenal mucosal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 261: G327-G331, 1991.[Abstract/Free Full Text]
  3. Bukhave K, Rask-Madsen J, Hogan DL, Koss MA, and Isenberg JI. Proximal duodenal prostaglandin E2 release and mucosal bicarbonate secretion are altered in patients with duodenal ulcer. Gastroenterology 99: 951-955, 1990.[ISI][Medline]
  4. Charney AN, Egnor RW, Alexander-Chacko JT, Zaharia V, Mann EA, and Giannella RA. Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice. Am J Physiol Gastrointest Liver Physiol 280: G216-G221, 2001.[Abstract/Free Full Text]
  5. Chaudhuri AG and Ganguly U. Evidence for stimulation of the inositol triphosphate Ca2+ signaling system in rat enterocytes by heat stable enterotoxin of Esherichia coli. Biochim Biophys Acta 1267: 131-133, 1995.[CrossRef][ISI][Medline]
  6. Closs EI, Enseleit F, Koesling D, Pfeilschifter JM, Schwarz PM, and Forstermann U. Coexpression of inducible NO synthase and soluble guanylyl cyclase in colonic enterocytes: a pathophysiologic signaling pathway for the initiation of diarrhea by gram-negative bacteria? FASEB J 12: 1643-1649, 1998.[Abstract/Free Full Text]
  7. Cohen MB, Hawkins JA, and Witte DP. Guanylin mRNA expression in human intestine and colorectal adenocarcinoma. Lab Invest 78: 101-108, 1998.[ISI][Medline]
  8. Cohen MB, Witte DP, Hawkins JA, and Currie MG. Immunohisto-chemical localization of guanylin in the rat small intestine and colon. Biochem Biophys Res Commun 209: 803-808, 1995.[CrossRef][ISI][Medline]
  9. Fan X, Wang Y, London RM, Eber SL, Krause WJ, Freeman RH, and Forte LR. Signaling pathways for guanylin and uroguanylin in the digestive, renal, central nervous, reproductive, and lymphoid systems. Endocrinology 138: 4636-4648, 1997.[Abstract/Free Full Text]
  10. Field M. Cruising the villus-to-crypt axis: the role of cyclic 3',5'-guanosine monophosphate in duodenal bicarbonate secretion. Gastroenterology 111: 1760-1763, 1996.[ISI][Medline]
  11. Flemstrom G and Jedstedt G. Stimulation of duodenal mucosal bicarbonate secretion in the rat by brain peptides. Gastroenterology 97: 412-420, 1989.[ISI][Medline]
  12. Forte LR. Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology. Reg Peptides 81: 25-39, 1999.[CrossRef][ISI][Medline]
  13. Guba M, Kuhn M, Forssmann WG, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: 1558-1568, 1996.[ISI][Medline]
  14. Hogan DL, Ainsworth MA, and Isenberg JI. Review article: gastroduodenal bicarbonate secretion. Aliment Pharmacol Ther 8: 475-488, 1994.[ISI][Medline]
  15. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky DM, and Ainsworth MA. Acid-stimulated duodenal bicarbonate secretion involves a CFTR-mediated transport pathway in mice. Gastroenterology 113: 533-541, 1997.[ISI][Medline]
  16. Holm M, Johansson B, Pettersson A, and Fandriks L. Acid-induced duodenal mucosal nitric oxide output parallels bicarbonate secretion in the anaesthetized pig. Acta Physiol Scand 162: 461-468, 1998.[CrossRef][ISI][Medline]
  17. Holm M, Johansson B, Von Bothmer C, Jonson C, Pettersson A, and Fandriks L. Acid-induced increase in duodenal mucosal alkaline secretion in the rat involves the L-arginine/NO pathway. Acta Physiol Scand 161: 527-532, 1997.[CrossRef][ISI][Medline]
  18. Howue KM, Pal A, Nair GB, Chattopadhyay S, and Chakrabarti MK. Evidence of calcium influx across the plasma membrane depends upon the initial rise of cytosolic calcium with activation of IP(3) in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-O1. FEMS Microbiol Lett 196: 45-50, 2001.[CrossRef][ISI][Medline]
  19. Huott PA, Liu W, McRoberts JA, Giannella RA, and Dharmsathaphorn K. Mechanism of action of Escherichia coli heat stable enterotoxin in a human colonic cell line. J Clin Invest 82: 514-523, 1988.[ISI][Medline]
  20. Isenberg JI, Ljungstrom M, Safsten B, and Flemstrom G. Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl-- exchange and Na- cotransport. Am J Physiol Gastrointest Liver Physiol 265: G677-G685, 1993.[Abstract/Free Full Text]
  21. Joo NS, London RM, Kim HD, Forte LR, and Clarke LL. Regulation of intestinal Cl- and secretion by uroguanylin. Am J Physiol Gastrointest Liver Physiol 274: G633-G644, 1998.[Abstract/Free Full Text]
  22. Keely SJ, Uribe JM, and Barrett KE. Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion. J Biol Chem 273: 27111-27117, 1998.[Abstract/Free Full Text]
  23. Krause WJ, Cullingford GL, Freeman RH, Eber SL, Richardson KC, Fok KF, Currie MG, and Forte LR. Distribution of heat-stable enterotoxin/guanylin receptors in the intestinal tract of man and other mammals. J Anat 184: 407-417, 1994.[ISI][Medline]
  24. Mann EA, Jump ML, Wu J, Yee E, and Giannella RA. Mice lacking the guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion. Biochem Biophys Res Commun 239: 463-466, 1997.[CrossRef][ISI][Medline]
  25. Markert T, Vaandrager AB, Gambaryan S, Pohler D, Hausler C, Walter U, De Jonge HR, Jarchau T, and Lohmann SM. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. J Clin Invest 96: 822-830, 1995.[ISI][Medline]
  26. Montrose MH, Keely SJ, and Barrett KE. Electrolyte secretion and absorption: small intestine and colon. In: Textbook of Gastroenterology, edited by Yamada T, Alpers DH, Owyang C, Powell DW, and Silverstein FE. Philadelphia, PA: Lippincott, 1999, p. 321-355.
  27. Moor AN and Fliegel L. Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways. J Biol Chem 274: 22985-22992, 1999.[Abstract/Free Full Text]
  28. Pfeifer A, Aszaodi A, Seidler U, Ruth P, Hofmann F, and Feassler R. Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science 274: 2082-2086, 1996.[Abstract/Free Full Text]
  29. Pratha VS, Hogan DL, Martensson B, Bernard J, Zhou R, and Isenberg JI. Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis. Gastroenterology 118: 1051-1060, 2000.[ISI][Medline]
  30. Robey RB, Ruiz OS, Espiritu DJ, Ibanez VC, Kear FT, Noboa OA, Bernardo AA, and Arruda JA. Angiotensin II stimulation of renal epithelial cell Na/ cotransport activity: a central role for Src family kinase/classic MAPK pathway coupling. J Membr Biol 187: 135-145, 2002.[CrossRef][ISI][Medline]
  31. Schulz S, Green CK, Yuen PS, and Garbers DL. Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell 63: 941-948, 1990.[ISI][Medline]
  32. Scott RO, Thelin WR, and Milgram SL. A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. J Biol Chem 277: 22934-22941, 2002.[Abstract/Free Full Text]
  33. Seidler U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans DF, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent secretion. J Physiol 505: 411-423, 1997.[Abstract]
  34. Short DB, Trotter KW, Reczek D, Kreda SMBA, Boucher RC, Stutts MJ, and Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273: 19797-19801, 1998.[Abstract/Free Full Text]
  35. Soh JW, Mao Y, Kim MG, Pamukcu R, Li H, Piazza GA, Thompson WJ, and Weinstein IB. Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun NH2-terminal kinase 1. Clin Cancer Res 6: 4136-4141, 2000.[Abstract/Free Full Text]
  36. Soh JW, Mao Y, Liu L, Thompson WJ, Pamukcu R, and Weinstein IB. Protein Kinase G Activates th JNK1 Pathway via Phosphoylation of MEKK1. J Biol Chem 276: 16406-16410, 2001.[Abstract/Free Full Text]
  37. Swenson ES, Mann EA, Jump ML, Witte DP, and Giannella RA. The guanylin/STa receptor is expressed in crypts and apical epithelium throughout the mouse intestine. Biochem Biophys Res Commun 225: 1009-1014, 1996.[CrossRef][ISI][Medline]
  38. Vaandrager AB, Bot AG, De Vente J, and De Jonge HR. Atriopeptins and Escherichia coli enterotoxin STa have different sites of action in mammalian intestine. Gastroenterology 102: 1161-1169, 1992.[ISI][Medline]
  39. Vaandrager AB and De Jonge HR. Effect of cyclic GMP on intestinal transport. Adv Pharmacol 26: 253-283, 1994.[Medline]
  40. Vaandrager AB, Travis SP, Welsh MJ, and De Jonge HR. Expression of type II cGMP-dependent protein kinase, a CFTR activator, in human bronchial epithelial cells. Pediatric Pulmonology Suppl 17, 239-240. 1998.
  41. Watts BA3, and Good DW. ERK mediates inhibition of Na+/H+ exchange and absorption by nerve growth factor in MTAL. Am J Physiol Renal Physiol 282: F1056-F1063, 2002.[Abstract/Free Full Text]