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Am J Physiol Gastrointest Liver Physiol 291: G650-G657, 2006. First published May 4, 2006; doi:10.1152/ajpgi.00376.2005
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MUCOSAL BIOLOGY

Carbachol increases Na+-HCO3– cotransport activity in murine colonic crypts in a M3–, Ca2+/calmodulin-, and PKC-dependent manner

O. Bachmann, D. Reichelt, B. Tuo, M. P. Manns, and U. Seidler

Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany

Submitted 12 August 2005 ; accepted in final form 25 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Na+-HCO3– cotransporter (NBC) mediates HCO3– import into the colonocyte via its pNBC1 isoform. Whereas renal kNBC1 is inhibited by increased cAMP levels, pNBC1 is stimulated. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from the murine proximal colon by Ca2+ chelation and loaded with the pH-sensitive dye 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein. Na+-HCO3– cotransport activity was calculated from the dimethylamiloride-insensitive (500 µM) intracellular pH recovery from an acid load in the presence of CO2-HCO3– and the intracellular buffering capacity. Carbachol strongly increased Na+-HCO3– cotransport activity compared with control rates. Ca2+ chelation with BAPTA-AM, blockade of the M3 subtype of muscarinergic receptors with 4-diphenylacetoxy-N-methylpiperidine methiodide, and inhibition of Ca2+/calmodulin kinase II with KN-62 all caused significant inhibition of the carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Gö-6976 and Gö-6850 significantly reduced the carbachol effect, which may be related to the unique NH2-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that NBC in the murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transductions pathways are distinct from those involved in the cholinergic activation of renal NBC1.

colon; anion secretion; ion transport; intestinal epithelium

INTESTINAL HCO3– secretion is stimulated by a variety of agonists, including cholinergic stimulation (42, 44). The rate-limiting steps for HCO3– secretion are intracellular CO2 hydration and basolateral uptake, which are mediated by various carbonic anhydrase isoforms (20) and the intestinal Na+-HCO3– cotransporter (NBC)1 subtype pNBC1 (6, 20, 24), respectively. We (4) have recently shown that in murine colonic crypts, NBC is directly activated by an increase in cAMP levels, but nothing is known about the influence of cholinergic stimulation on NBC activity in the native intestine.

pNBC1 is 93% homologous to the renal NBC1 subtype kNBC1, but there is to date a substantial body of evidence for differences in stochiometry, regulation, and physiological function (43, 46). Whereas kNBC1 exports Na+ and HCO3– with a 3:1 ratio, pNBC1 serves to uptake both ions in the intestinal epithelium and pancreas (4, 17). Furthermore, the NH2 terminus of pNBC1 contains consensus phosphorylation sites for PKA, PKC, and casein kinase II, which kNBC1 lacks (2). Whereas renal Na+-HCO3– cotransport is inhibited by an increase in intracellular cAMP, we (4) have recently demonstrated cAMP-dependent stimulation of this transport process in murine colonic crypts. Subsequently, Gross et al. (16) investigated the structure-function relationship for these contrasting findings of pNBC1 and reported that PKA-dependent phosphorylation of a common serine residue (Ser982 for kNBC1 and Ser1026 for pNBC1) shifts the stochiometry from 3:1 to 2:1 when this subtype is transfected into a renal cell line, whereas 8-bromo-cAMP leads to increased activity of NBC without stoichiometry changes in mouse pancreatic cells. This indicates that the regulatory properties of the NBC1 subtypes depend on primary structure but also on cell type.

Cholinergic regulation of the NBC has been studied in the kidney, where this transport mechanism is stimulated by carbachol (12, 35, 38). In primary cultures of rabbit proximal tubules, this stimulatory effect was reversible by Ca2+ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC depletion, M1 receptor blockade (38), and the nitric oxide inhibitor N-nitro-L-arginine methyl ester (39). Subsequently, the same group reported involvement of tyrosine kinase activity in this process and found a role for the Src family of nonreceptor tyrosine kinases (35), which also mediate the stimulation of Na+/H+ exchanger (NHE) isoform 3 and NBC during acute acidosis to enhance HCO3– reabsorption (49).

Na+-HCO3– cotransport fulfills several important tasks in different segments of the intestinal tract. In the duodenum, it serves to import HCO3– destined for secretion as part of the gastroduodenal barrier against peptic damage (20, 30). In the duodenum and colon, Na+-HCO3– cotransport and anion exchange work in concert as an alternative Cl– uptake pathway, explaining in part the residual Cl– secretory activity in Na+-K+-2C1– isoform 1-null intestinal tissues (21, 51).

The role of cholinergic stimuli in the case of the intestinal NBC is not clearly defined. In guinea pig pancreatic interlobular ducts, cholinergic secretagogues lead to the activation of a Na+-dependent, N-methyl-N-isobutylamiloride-insensitive basolateral HCO3– uptake pathway, consistent with enhanced Na+-HCO3– cotransport activity during cholinergic stimulation of pancreatic fluid and electrolyte secretion (19). The aim of the present study was to assess the existence of cholinergic stimulation of the intestinal NBC and the involved signal transduction pathways.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Materials. BSA (cell culture grade) was obtained from Paesel und Lorei (Frankfurt, Germany), and 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF-AM) as well as BAPTA-AM were from Molecular Probes (Leiden, The Netherlands). 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), 1-[N,o-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö-6976), and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Gö-6850; bisindolylmaleimide I) were purchased from Calbiochem (Bad Soden, Germany). All other chemicals were either obtained from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available.

Animal breeding. C57/BL6 mice were raised in the animal facility of the Medical School of Hannover under standardized light and climate conditions and had access to water and chow ad libitum. Animal experiments followed approved protocols at the Medical School of Hannover and the local authorities for the regulation of animal welfare (Regierungspräsidium).

Preparation of colonic crypts. Murine colonic crypts were isolated as previously described (4). Briefly, animals were killed by CO2 narcosis after cervical dislocation. After an abdominal midline incision was made, a 3-cm segment of the proximal colon was excised, washed through thoroughly with ice-cold buffer A [composition (in mM) 120 NaCl, 14 HEPES, 7 Tris, 3 KH2PO4, 2 K2HPO4, 1.2 MgSO4, 1.2 Ca2+-gluconate, and 20 glucose; pH 7.4] gassed with 100% O2, everted, filled with EDTA-containing solution [composition (in mM) 127 NaCl, 5 KCl, 1 MgCl2, 5 Na-pyruvate, 10 HEPES, and 5 EDTA with 1% BSA; pH 7.4], and incubated in the same solution at 37°C with gentle mixing. After 15 min, crypts were separated from the submucosal tissue by applying shear stress, harvested by low-speed centrifugation, quickly immersed in ice-cold buffer A gassed with 100% O2, and kept on ice until use. Viability testing was carried out using trypan blue exclusion.

Intracellular pH measurements. Crypts were loaded with 5 µM BCECF in buffer A at room temperature followed by a 30-min washout period in buffer A. Subsequently, 50 µl of the crypt suspension were pipetted on a glass coverslip. After 2–3 min of sedimentation, crypts were fixed on the coverslip with a polycarbonate membrane (25 mm diameter, pore size 3 µm; Osmonics, Minnetonka, MN) in a custom-made perfusion chamber (Zentrale Forschungswerkstätten of the Medizinische Hochschule Hannover, Hannover Germany), mounted onto the heated stage of an inverted microscope (Zeiss Axiovert 200, Carl Zeiss, Jena, Germany), and perfused with prewarmed (37°C) 95% O2-5% CO2-gassed HCO3–-containing buffers (except the calibration buffers) following the appropriate protocol [in buffer B, 20 mM NaCl of buffer A was replaced by 20 mM NaHCO3; in buffer C, 40 mM NaCl of buffer B was replaced by 40 mM NH4Cl; and in buffer D, NaCl and NaHCO3 of buffer B were replaced by tetramethylammonium chloride (TMA-Cl) and choline-HCO3–, respectively]. Images were digitized every 2–30 s with a cooled charge-coupled device camera (CoolSnap ES, Roper Scientific, Ottobrunn, Germany) using Metafluor software (Universal Imaging, Downington, PA) during exposure of cells to alternating 440- and 490-nm light from a monochromator (Visichrome, Visitron Systems, Puchheim, Germany) with a 515-nm DCXR dichroic mirror and a 535-nm barrier filter (Chroma Technology, Rockingham, VT) in the emission pathway. Calibration of the 440-to-490-nm ratio was performed as described previously (4) for pH values 6.6 and 7.4 using the high K+-nigericin method [solution composition (in mM) 100 K-gluconate, 40 KCl, 7 HEPES, 1.2 Ca2+-gluconate, 1.2 MgSO4, and 20 glucose, with 10 µM nigericin; pH 7.4 or 6.6, gassed with 100% O2].

At the end of each experiment, regions of interest (ROIs) were selected covering the lower two-thirds of the crypts, which were previously found to contain pNBC1 by in situ hybridization (4), and the 440-to-490-min time course was reproduced from the stored images after background substraction, followed by conversion to intracellular pH (pHi) values using Microsoft Excel (Microsoft, Redmond, WA).

Preparation of isolated colonic mucosa. To measure colonic HCO3– secretion, mice were killed by brief narcosis with 100% CO2 and cervical dislocation. The abdomen was opened, and the colon was removed and placed in ice-cold isoosmolar mannitol and indomethacin (1 µmol/l) solution (to suppress trauma-induced prostaglandin release). The colon was opened along the mesenteric border, and the proximal colon was stripped of external serosal and muscle layers by sharp dissection. The proximal colonic mucosa was mounted between two chambers with an exposed area of 0.625 cm2 and placed in an Ussing chamber. Parafilm "O" rings were used to minimize edge damage to the tissue where it was secured between the chamber halves. The mucosal side was bathed with unbuffered HCO3–-free modified Ringer solution circulated by a gas lift with 100% O2. The serosal side was bathed with modified buffered Ringer solution (pH 7.4) containing 22 mmol/l HCO3– and gassed with 95% O2-5% CO2. Each bath contained 10.0 ml of the respective solution maintained at 37°C by a heated water jacket. Experiments were performed under continuous short-circuited conditions to maintain the electrical potential difference at zero except for a brief period (<2 s) at each time point when the open circuit potential difference was measured. Luminal pH was maintained at 7.40 by the continuous infusion of 1 mmol/l HCl under the automatic control of a pH stat system (PHM290, pH-Stat Controller, Radiometer, Copenhagen, Denmark). The volume of the titrant infused per unit time was used to quantitate HCO3– secretion. These measurements were recorded at 5-min intervals. The rate of luminal HCO3– secretion is expressed as micromoles per centimeter squared per hour. Transepithelial short-circuit current (Isc; reported as µeq·cm–2·h–1) was measured via an automatic voltage clamp (Voltage-Current Clamp, EVC-4000, World Precision Instruments, Berlin, Germany). After a 40-min measurement of basal parameters, carbachol (10–4 M) was added to the serosal side of the tissue in the Ussing chambers. Changes in colonic HCO3– secretion and Isc during the 40-min period ensuing after the addition of stimulants were determined.

Statistics and calculation of proton fluxes. Results are given in means ± SE. Proton fluxes were calculated by multiplying the initial steep pHi slope after the readdition of Na+, which was determined by regression analysis, with the total buffering capacity at the initial pHi, including the intrinsic buffering capacity (betai) and, in addition, the CO2-dependent buffering capacity for CO2-HCO3–-containing solutions (4). Statistical significance was determined using ANOVA with Tukey's honestly significant difference test as a post hoc test for multiple comparisons and the Scheffe procedure for complex comparisons, respectively, and Student's t-test in its unpaired form for pairwise comparisons. For SE calculation and statistics, n = 1 was defined as the mean of values from the ROIs of one experiment.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional dissection of Na+-HCO3– cotransport and Na+/H+ antiport activity in isolated murine colonic crypts. The cryptal steady-state pH was 7.41 ± 0.02 in the presence of CO2-HCO3– and 7.44 ± 0.03 in its absence. After a NH4+ prepulse, pHi recovery resulted in higher flux rates in the presence than in the absence of CO2-HCO3–. The acid-activated HCO3–- and Na+-dependent flux was calculated as 3.98 mM/min or 17.2% of the total flux rate in CO2-HCO3–, and we (4) have previously shown that this flux rate is highly sensitive to the NBC inhibitory compound S0859. To block Na+/H+ exchange, which would likely interfere with the experiments regarding NBC regulation due to different regulatory properties, the subsequent measurements were carried out in the presence of a 700 µM concentration of the Na+/H+ exchange inhibitor dimethylamiloride (DMA). This compound was added after the NH4+ prepulse and did not alter pHi during a short incubation period (2 min). Subsequently, the readdition of Na+ led to pHi recovery corresponding to a flux rate of 5.43 ± 0.42 mM/min, which was mostly mediated by Na+-HCO3– cotransport (4). In the absence of CO2-HCO3–, the recovery-associated flux rate was very low (Fig. 1). Overall, the calculated NBC activities were virtually identical in the absence and presence of DMA.


Figure 1
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  		Fig. 1. Demonstration of Na+-HCO3– cotransport in isolated murine colonic crypts. A: intracellular pH (pHi) recovery from an acid load occurred faster in CO2-HCO3–-buffered solutions. TMA-Cl, tetramethylammonium chloride. B: in the absence of CO2-HCO3–, the control flux rate (19.20 ± 1.27 mM/min) was reduced to 1.59 ± 0.12 mM/min after Na+/H+ exchange inhibition with 700 µM dimethylamiloride (DMA). In its presence, the control flux rate was higher (23.18 ± 1.29 mM/min, P < 0.05) and the residual flux after DMA, representing Na+-HCO3– cotransport activity, was also significantly higher than in O2 (5.43 ± 0.42 mM/min, P < 0.01 by Student's t-test, n = 10–15 experiments with 7–10 mice). C: the difference in flux rates between CO2-HCO3–-containing and CO2-HCO3–-free systems was not different in the presence and absence of DMA [not significant (n.s.) by ANOVA and Scheffe's procedure].

 
The high concentration of 700 µM DMA was necessary to sufficiently block NHE isoforms expressed in the crypt base (3). Although the fact that the difference between the flux rates in the presence and absence of CO2-HCO3– was similar in the presence and absence of DMA (Fig. 1C) strongly argues against NBC inhibition by this compound, as suggested in earlier experiments by Rajendran et al. (34), we sought to further substantiate this finding by experiments with the Na+/H+ exchange inhibitor HOE642 (3). This compound dose dependently blocks NHE1 and NHE2, and we have previously confirmed the dose of 50 µM for NHE1 and NHE2 inhibition found in transfected fibroblasts (41) for use in our experimental setup (3). HOE642 (50 µM) lowered the recovery-associated flux rate in the absence of CO2-HCO3– to a small remaining flux, which corresponds possibly to residual NHE3 activity or other, unidentified transport mechanisms. This flux rate together with the one in the presence of CO2-HCO3– and DMA was equivalent to the flux rate in the presence of CO2-HCO3– and 50 µM HOE642, further substantiating the notion that 700 µM DMA did not significantly alter Na+-HCO3– cotransport rates (Fig. 2).


Figure 2
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  		Fig. 2. Proton flux rates in the presence of DMA versus HOE642. HOE642 at a concentration of 50 µM inhibited the Na+/H+ exchanger isoforms NHE1 and NHE2 but not NHE3 (bar 4). In the presence of this compound and of CO2-HCO3– (bar 3), the proton flux rate during recovery from an acid load was significantly higher than with DMA and CO2-HCO3– [bar 1, Na+-HCO3– cotransporter (NBC)], demonstrating additional activity of NHE3 and possibly other, unidentified transport mechanisms (6.16 ± 0.19 vs. 4.69 ± 0.55 mM/min, P < 0.05 by Student's t-test). Bars 1 and 4 are equivalent to bar 3, indicating that DMA did not directly change NBC activity (n = 6–8 experiments with 3–5 mice).

 
Carbachol stimulates Na+-HCO3– cotransport. Carbachol enhances intestinal anion secretion and Na+-HCO3– cotransport activity in isolated cells from the rabbit proximal tubule (38), and earlier work in the guinea pig pancreas has demonstrated stimulation of Na+-dependent basolateral HCO3– uptake by cholinergic secretagogues (19). Given the contrasting effects of cAMP-dependent stimulation on the renal and intestinal NBC1 subtypes, we wanted to test the effect of cholinergic stimulation with carbachol on Na+-HCO3– cotransport rates at a cellular level in our colonic crypt model. As shown in Fig. 3A, carbachol (100 µM) strongly increased DMA-insensitive, Na+- and HCO3–-dependent pHi recovery from an acid load compared with the control. Figure 3B shows the corresponding proton/base flux rates compared with the stimulation caused by forskolin.


Figure 3
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  		Fig. 3. Effect of carbachol on acid-induced proton flux rates in the presence and absence of CO2-HCO3–. A: preincubation with carbachol (100 µM) markedly accelerated pHi recovery from an acid load in the presence of CO2-HCO3–. B: this resulted in flux rates that were significantly higher than under control conditions (10.25 ± 0.72 vs. 5.43 ± 0.42 mM/min, P < 0.05 by Student's t-test, n = 14–16 experiments with 6–8 mice). For comparison, stimulation of NBC with forskolin is shown (8.20 ± 0.37 vs. 5.07 ± 0.70 mM/min, P < 0.05 by Student's t-test, n = 6–7 experiments with 4 mice). In the absence of CO2-HCO3–, carbachol caused no change in acid-induced flux rates (1.59 ± 0.12 vs. 1.92 ± 0.70 mM/min, n.s. by Student's t-test, n = 6–7 experiments from 3–4 mice).

 
To verify that the entire stimulatory effect of carbachol is attributable to NBC, we conducted experiments in CO2-HCO3–-free solution. We have previously found inhibition of all NHE isoforms by 700 µM DMA, and NHE3 has been reported to be inhibited by carbachol in rabbit ileal brush border (26), but this compound could possibly activate unidentified, HCO3–-independent pHi recovery pathways. However, the recovery-associated flux rate after carbachol was not significantly different from the control (Fig. 3C).

Effect of Ca2+ chelation, muscarinic receptor blockade, and Ca2+/calmodulin-dependent kinase II inhibition on carbachol-associated NBC stimulation. Ca2+ chelation with BAPTA (50 µM) alone did not change the control flux rate, but preincubation for 10 min with BAPTA-AM completely inhibited the carbachol-associated NBC stimulation (Fig. 4A). In human duodenal enterocytes, carbachol causes an increase in intracellular Ca2+, which can be prevented by blocking the muscarinergic receptor subtype M3 with 4-DAMP, whereas M1 and M2 blockade have no effect (8, 27). Preincubation with 4-DAMP (100 nM) and subsequent carbachol stimulation yielded flux rates not significantly different from the unstimulated control (Fig. 4B), indicating that the M3 receptor is necessary to transmit the signal. In the kidney, inhibition of Ca2+/calmodulin-dependent kinase (CaMK) with W-13 prevents cholinergic NBC stimulation (38), whereas calmodulin itself is inhibitory (37). The CaMKII isoform of this enzyme has been demonstrated in the basolateral and apical membranes of the rat jejunum (48), and its introduction into the intestinal cell line T84 induces Cl– secretion (55). To clarify the role of this enzyme in carbachol-dependent NBC activation in the colon, we therefore studied the effect of CaMKII inhibitor KN-62 (10 µM for 10 min). This compound completely abolished the stimulatory action of carbachol.


Figure 4
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  		Fig. 4. Effect of Ca2+ chelation, M3 receptor blockade, and Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition on carbachol-induced stimulation of NBC. A: in the presence of 50 µM BAPTA-AM, acid-induced proton flux rates in the presence of DMA and CO2-HCO3– were not different with and without carbachol (5.85 ± 0.66 vs. 5.36 ± 0.52 mM/min, n.s. by Student's t-test). B: after blockade of M3 receptors by a 10-min preincubation with 100 nM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), NBC-mediated proton flux was not significantly stimulated by carbachol (4.41 ± 0.63 vs. 4.23 ± 0.32 mM/min). This was also observed for CaMKII inhibition with 10 µM KN-62 (3.82 ± 0.54 vs. 4.80 ± 0.60 mM/min). #n.s. by ANOVA and Tukey's HSD test; n = 6–9 experiments with 4–5 mice.

 
Effect of PKC inhibition during cholinergic stimulation of Na+-HCO3– cotransporter activity. pNBC1 possesses a consensus site for PKC-dependent phosphorylation, which is absent in kNBC1 (2). To examine whether PKC is involved in the regulation of NBC activity, we preincubated colonic crypts with Gö-6976, which blocks the conventional PKC isoform, and with Gö-6850, which blocks both conventional and novel isoforms (47). Whereas Gö-6976 caused partial inhibition of the carbachol-induced stimulation of NBC activity, Gö-6850 reduced the flux rates to control values (Fig. 5).


Figure 5
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  		Fig. 5. Effect of PKC inhibition during cholinergic stimulation of NBC acitivity. A: pharmacological inhibition of PKC isoforms with Gö-6976 (5 µM) and Gö-6850 (5 µM) blocked the stimulatory effect of carbachol partially and completely, respectively (6.55 ± 2.24 and 3.78 ± 0.61 vs. 9.00 ± 1.91 mM/min). *P < 0.05 and **P < 0.01 by ANOVA and Tukey's HSD test; n = 5–11 experiments with 3–5 mice. B: PKC activation with the phorbol ester PMA (10–7 M for 10 min) did not produce significant changes in NBC activity compared with control (4.17 ± 0.73 mM/min, n.s. by Student's t-test, n = 5–6 experiments with 3–4 mice).

 
Ussing chamber experiments. Because the effect of carbachol on colonic HCO3– secretion has not been reported, we studied carbachol-stimulated Isc and the HCO3– secretory response in the stripped proximal colon. Compared with basal values, carbachol caused an increase in Isc and the HCO3– secretory rate. These data indicate stimulation of Isc and HCO3– in the murine colonic mucosa by cholinergic stimulation (Fig. 6).


Figure 6
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  		Fig. 6. Effect of cholinergic stimulation on short-circuit current (Isc) and HCO3– secretory rates in the Ussing chamber. A: HCO3– secretory rates in isolated stripped murine colonic mucosa in response to carbachol. JHCO3–, HCO3– flux rate. The basal rate (1.33 ± 0.14 µeq·cm–2·h–1) was stimulated by 0.38 µeq·cm–2·h–1. B: Isc was 4.15 ± 0.95 µeq·cm–2·h–1 before and 8.01 ± 1.47 µeq·cm–2·h–1 after carbachol stimulation. *P < 0.05 by Student's t-test; n = 9 experiments in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we investigate the effect of carbachol, an important agonist of anion secretion in the intestinal tract (Refs. 10, 42, and 44; for reviews, see Refs. 6 and 23), on basolateral Na+-HCO3– cotransport, which not only mediates the import of HCO3– destined for secretion (20) but can also serve as an alternative uptake pathway for Cl– (21, 51). Using isolated conolic crypts (4), we were able to show at a cellular level that NBC is activated by carbachol via the muscarinic receptor subtype M3. Furthermore, we demonstrated the involvement of Ca2+ and CaMKII as well as several PKC isoforms.

In our functional studies, we measure total NBC activity. However, we have good reasons to assume that this activity is largely if not completely mediated by the pNBC1 subtype of the electrogenic NBC1. We previously showed by semiquantitative PCR that the intestinal/pancreatic splice variant of the SLC4A gene, pNBC1, is expressed at much higher levels in the intestine than kNBC1 (43) and is strongly enriched in colonic crypts compared with the entire colonic mucosa (4). The electroneutral, amiloride-sensitive isoform NBC3 (NBCn1, SLC4A7) (32) was also detected, but to a markedly lower extent (4). In contrast to those for NBC1, the expression levels of NBC3 were higher in colonic mucosal scrapings than in isolated crypts, pointing to a subepithelial expression of NBC3, possibly in muscular tissue (4). The expression of closely related NBC2, which was initially cloned from the eye (18), was very low in the intestine (20, 36). The studies concerning NBC4 variants (29, 33, 40, 56) have yielded controversial results, and no expression of the Na+-, HCO3–-, and Cl–-transporting proteins NDCBE1 and NCBE was found in the colon (15, 52). Data recently presented by Barmeyer et al. (5) in abstract form suggest the expression of an NBC3-like, amiloride-sensitive NBC in the rat proximal colon. However, amiloride-sensitive Na+-HCO3– cotransport does not play a major role in our experimental setup, because the difference between the pHi recovery rate in the presence and absence of CO2-HCO3– was the same with or without DMA. Taken together, we cannot entirely exclude that additional NBCs may be functional in murine colonic crypts, but the prevailing evidence suggests that pNBC1 is an important mediator of colonic Na+-HCO3– cotransport.

The renal and the intestinal/pancreatic NBC1 subtypes display major differences in physiological function, stoichiometry (which relates to transport direction), and regulation. Whereas renal NBC1 has been studied more extensively (for a review, see Ref. 1), much less is known about the intestinal/pancreatic subtype. We and others (4, 16) have recently shown contrasting effects of increased intracellular cAMP on renal and intestinal/pancreatic NBC1 consistent with structural differences. However, cholinergic stimulation has, so far, only been investigated in the kidney, where carbachol caused stimulation of the cotransporter with a 90% activity increase at 10–5 M (38) that could be inhibited by M1 muscarinic receptor blockade. In contrast to this, our results demonstrate that the carbachol effect on colonic NBC is mediated via the M3 subtype, which is consistent with the findings of others (8, 27) who have studied cholinergic signaling in intestinal tissues. In the kidney, NBC1 and NHE3 work in tandem to accomplish HCO3– reabsorption. In most experimental models of intestinal anion secretion, cholinergic stimulation by carbachol has been shown to evoke a Cl– and HCO3– secretory response (42, 44), and NHE3 is inhibited by increased intracellular Ca2+ levels in heterologous expression systems and intestinal epithelial cells (22, 26). In this setting, activation of NBC1 as part of the anion secretory pathway is expected to coincide with the simultaneous inhibition of NHE3 as a mediator of electroneutral NaCl absorption.

NBC has been shown to be involved in anion secretion in both the intestine and pancreas (19, 20, 45). To date, the effect of cholinergic stimulation on intestinal or pancreatic Na+-HCO3– cotransport has not been studied directly. Ishiguro et al. (19) measured luminal alkalinization of isolated guinea pig pancreatic intralobular ducts and described carbachol-stimulated HCO3– secretion after removal of serosal Na+ but not after the addition of N-methyl-N-isobutylamiloride. In the present study, however, we measure NBC in native colonocytes and showed its activation by carbachol. Instead of directly activating NBC, this compound could theoretically lead to anion secretion, and NBC activation could occur secondarily to secretion-associated changes like volume or anion loss. However, our experimental setup virtually rules this out, because we measured NBC activity after a NH4+ prepulse and preincubation in Na+-free, DMA-containing solution. Several groups (25, 31, 54, 57) have found strong inhibition of anion secretion after exposure to NH4+ in different secretory epithelia. In some of these studies, the inhibitory effect could be demonstrated as long as 30 min after NH4Cl exposure (25, 54). To verify this for carbachol stimulation in the murine colon, we measured Isc in Ussing chambers using the murine colonic mucosa after a NH4Cl prepulse and DMA preincubation. Indeed, this resulted in a strong reduction of the Isc response to carbachol compared with the control (data not shown).

Binding of carbachol to muscarinic receptors stimulates the phospholipase C pathway, leading to the production of inositol-1,4,5-trisphosphate [I(1,4,5)P3] and diacylglycerol. This results in an increase in the intracellular Ca2+ concentration (8, 10) and/or activation of PKC (42). In our study, Ca2+ chelation with BAPTA inhibited the carbachol-mediated increase in NBC activity. To investigate the further function of Ca2+, we inhibited CaMKII, which is localized in intestinal tissues (48), with KN-62. This kinase promotes anion secretion when introduced into the T84 intestinal cell line (55) and can mediate the activation of Cl– secretion by Ca2+-dependent Cl– channels in respiratory epithelia (14). On the other hand, it has been shown to inhibit Na+ absorption by Na+/H+-exchange in the rabbit ileum (9). Consistent with a role of CaMKII during intestinal anion secretion, its inhibition strongly reduced carbachol-mediated NBC stimulation in our model.

Carbachol can also lead to PKC activation, and pNBC1 contains a unique putative consensus site for PKC-dependent phosphorylation, which is absent in the kNBC1 sequence (2). Ruiz et al. (38) have shown a reduction of carbachol-induced NBC stimulation in the rabbit kidney by PKC depletion, which points to a modulating role of PKC during cholinergic signaling. In their study (28) on the other important intestinal anion uptake pathway, NKCC1, Matthews et al. found PKC-dependent downregulation of NKCC1 expression and NKCC function, which was explained by increased endocytosis of the transport system. PMA caused prompt activation of the novel PKC-{epsilon} isoform, followed by late activation of the conventional PKC-{alpha} isoform, and opposing effects on basolateral membrane dynamics have been reported (47). Tuo et al. (50) described potentiation of cAMP-stimulated duodenal HCO3– secretion by activation of PKC-{epsilon} without modification of basal secretion. In our study, inhibition of conventional PKC with Gö-6976 and both conventional and novel PKCs with Gö-6850, respectively, partially and completely blocked the stimulatory effect of carbachol, indicating the involvement of both conventional and novel PKCs during carbachol exposure.

One remaining question is whether the different intracellular signaling mechanisms, which appear to be important for cholinergic activation of intestinal NBC, namely, Ca2+, CaMKII, and PKC, represent overlapping control mechanisms or are activated subsequently. Because some PKC isoforms are Ca2+ dependent, inhibition of NBC stimulation by carbachol by Ca2+ chelation could be explained by impaired translocation and activation of PKC (11). However, we found that only the inhibition of both the conventional, Ca2+-dependent, and novel, Ca2+-independent PKC isoforms caused full inhibition of cholinergic NBC stimulation. The fact that blockade of CaMKII or PKC each completely reversed the carbachol effect strongly argues against redundant signaling and indicates that neither mechanism can compensate for the loss of the other. Therefore, these pathways could theoretically be part of a hierarchical signaling system or represent two necessary components at the level of the transporter; it is unknown whether intestinal NBC is actually phosphorylated by these kinases. Additional possible regulatory mechanisms are the formation of a regulatory complex with adapter proteins or vesicle traffic. Arruda and collegues (7, 53) have reported an interaction of PDZ domain-binding proteins with renal NBC; furthermore, subcellular redistribution and changes in cell surface expression, which are important for many ion transport processes, have been found to regulate renal NBC (13). Whether these mechanisms are involved in intestinal NBC regulation requires further investigation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Hochschulinterne Forschungsförderung of Hannover Medical School and by Deutsche Forschungsgemeinschaft Grants Ba2114/5-1, Se460/13-2, and Se 460/9-4/5.


   ACKNOWLEDGMENTS
 
The authors are indebted to Prof. Ingo Just (Institute of Toxicology, Hannover Medical School) for the use of his laboratory facilities. They also thank Dr. H.-J. Lang and Dr. J. Pünter from Aventis Pharma (Frankfurt, Germany) for the generous contribution of the HOE642 compound. This article includes experimental work by D. Reichelt in fulfillment of the requirements for her doctoral thesis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Seidler, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany (e-mail: seidler.ursula{at}mh-hannover.de)

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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 ABSTRACT
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