cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter subtype pNBC1

O. Bachmann1, H. Rossmann1, U. V. Berger2, W. H. Colledge3, R. Ratcliff3, M. J. Evans4, M. Gregor1, and U. Seidler1

1 Department of Internal Medicine, University of Tübingen, D-72076 Tübingen, Germany; 2 In Situ Hybridization Core Facility, Beth Israel Deaconess Medical Center, Boston, MA 02115; 3 Department of Physiology, University of Cambridge, Cambridge CB2 3EG; and 4 Cardiff School of Biosciences, Cardiff CF10 3U5, United Kingdom


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Basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rates were 5.07 ± 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activation was secondary to secretion-associated changes in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activation by forskolin. These studies establish the existence of an intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.

bicarbonate secretion; colon; ion transport; chloride secretion; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein


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ALL SEGMENTS OF THE INTESTINAL tract secrete HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and in all segments, its secretory rate is inhibited both by azetazolamide, which inhibits the hydration of CO2, and by stilbene derivatives, which inhibit several classes of anion transporters, among them all currently known isoforms of the recently discovered gene family of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters. Similar to the pancreas, functional data suggest that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is imported by Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport during cAMP-induced HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and that the basolateral uptake and/or intracellular generation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is the rate-limiting step for intestinal and pancreatic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (11, 17, 18, 42). Thus more information about the agonist-dependent regulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> contransport rates in the gastrointestinal (GI) tract is desirable to design pharmacological strategies to influence it during both excess intestinal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loss, as in diarrheal states, and insufficient secretion, as in cystic fibrosis.

After the cloning of the renal Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC1 in 1997 (29), other isoforms of this gene family were rapidly discovered. These were named either according to the sequence of their cloning [NBC1 (2, 28), NBC2 (15), NBC3 (4), and NBC4 (25)] or named NBC1 for the first electrogenic (28), NBCn1 for the first electroneutral NBC isoform (9), and NBCE for the Na+-dependent Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (38). In the rabbit intestine, we found the expression of several of these NBC isoforms, with the highest expression level for NBC1, followed by NBC2 (1 variant of which is NBCn1), and very low NBC3 levels (18, 30, 34).

In the meantime, we and others found that two NBC1 subtypes with different NH2-terminal ends exist. In the kidney, only the renal NBC1 (kNBC1) is expressed, whereas the intestinal/pancreatic subtype (pNBC1) is expressed more widely (2, 18). In the mouse colon, small amounts of kNBC1 are expressed in addition to pNBC1 (32). Interestingly, the cytoplasmic NH2 terminus of the pNBC1 subtype contains a highly conserved PKA consensus site, which is lacking in the kNBC1 subtype, (2, 18). Thus the structural basis for a direct cAMP-dependent regulation of transport activity exists. However, whether the observed effect of cAMP on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity is direct, or secondary to apical transport processes, has not yet been studied.

In intact intestinal epithelium, basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rates cannot be measured precisely. In search of a model in which both basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity and apical anion secretion could be directly assessed and manipulated, we remembered the high NBC1 expression levels that we had observed in rabbit proximal colon (18). We localized NBC1 in mouse proximal colon to the cryptal region by in situ hybridization and measured the expression levels of the electrogenic NBC1 and of NBCn1, an electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter whose expression has been described in the duodenum. We then used isolated colonic crypts to measure acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rates and the effect of increasing intracellular cAMP on cotransport activity. When a marked stimulation was observed, we assessed whether it was likely secondary to cAMP-induced apical anion secretion by measuring the effect of forskolin on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in crypts from CFTR -/- mice, which display no cell shrinkage indicative of secretion (6), and in normal crypts in the presence of K+-channel inhibition, which also inhibits anion secretion (22). The results suggest that cAMP activates colonic NBC independent of cAMP-induced changes in intracellular pH (pHi), anion secretion, or cell volume.


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Materials. S0859, an NBC1 inhibitor, was from Aventis (Frankfurt, Germany). This substance inhibits the heart/pancreatic NBC1 (at low sodium concentration) with an IC50 of 5.9 µM and does not affect NBC2/3 when expressed in Chinese hamster ovary cells (Willi Jansen, Aventis laboratories, unpublished observations). BSA (cell culture grade) was obtained from Paesel und Lorei (Frankfurt, Germany) and BCECF was obtained from Molecular Probes (Leiden, the Netherlands). All other chemicals were either obtained from Sigma (Deisenhofen, Germany) or from Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available.

Animal breeding and genotyping. For the experiments with control crypts, we used the CFTR +/+ mice that were generated during the breeding of a CFTR-deficient mice strain. Due to the increased postnatal death rate of the CFTR -/- mice, the CFTR +/+ littermates are much more abundant. For the experiments in which CFTR -/- and +/+ crypts were compared, the crypts were derived from littermates. The cystic fibrosis null mice (CFTRtm1Cam) had been established in the laboratories of R. Ratcliff, W. H. Colledge, and M. Evans and previously characterized (27). They were raised in the animal facility of the Department of Biochemistry in Tübingen and genotyped as previously described (33). The protocol for the animal study was approved by the local authorities for animal welfare (university veterinarian and Regierungspräsidium Tübingen).

Preparation of colonic crypts. After CO2 narcosis and cervical dislocation, a 3-cm segment of proximal colon was excised, washed through with ice-cold buffer A gassed with 100% O2 (in mM: 120 Na+, 7 K+, 1.2 Mg2+, 1.2 Ca2+, 120 Cl-, 14 HEPES, 7 Tris, 3 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 2 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.2 gluconate, 20 glucose, pH 7.4), everted, and filled with Ca2+-free buffer B (composition in mM: 133 Na+, 5 K+ 135 Cl-, 1 Mg2+, 5 pyruvate, 10 HEPES, 5 EDTA, 1% BSA, pH 7.4). The segment was placed into a tube filled with solution B and vibrated at 10 Hz at room temperature. At defined intervals, the solution was replaced and epithelial fragments were harvested by low-speed centrifugation. Early fractions contained surface cells, later fractions contained crypts, both easily identified under a microscope, and their viability was tested by trypan blue exclusion.

In situ hybridization of NBC1 in murine proximal colon. Nonradioactive in situ hybridization was performed as described (31), using two digoxigenin (DIG)-labeled cRNA probes that comprised nucleotides 143-2113 and 2234-3495 of rkNBC. Frozen sections (10 µm) were cut in a cryostat and captured onto Superfrost plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed and acetylated and hybridized at 68°C over three nights to the NBC probes (approximate concentration 100 ng/ml). Hybridized probe was visualized using alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche, Indianapolis, IN) and 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate (Kierkegard and Perry Laboratories, Gaithersburg, MD). Sections were rinsed several times in 100 mM Tris, 150 mM NaCl, 20 mM EDTA, pH 9.5, and placed on a coverslip with glycerol gelatin (Sigma, St. Louis, MO). Control sections were incubated in an identical concentration of the sense probe transcript.

Cloning of murine NBCn1 cDNA fragments. RNA was isolated from mouse tissue, and first-strand cDNA was synthesized as described previously (30). One described forward primer [5'-GGAACAGTCATGCTGGATA-3' (24)] and two heterologous reverse primers (5'-TGTTAGAGGTTGCCCAGCAAAC-3', 5'-AGGAGACATACAGGCACAGTATAGG-3', deduced from published sequence information) were chosen for cloning NBCn1 cDNA fragments from murine colon. Amplification by touchdown PCR, cloning of a 1104- and a 991-bp PCR product, and sequencing was performed as described (30). Both PCR products were ~350 nucleotides smaller than expected, and sequencing revealed the lack of a 369-bp fragment (compared with human and rat sequence). A homologous primer pair (forward primer: 5'-AAGGGAGAACGTCAGAGAAGC-3', reverse primer: 5'-GATTACAGGAGACATACAGGC ACA-3') was deduced from the mouse sequence. PCR resulted in one major band at the expected size of 890 bp. Its molecular identity (NBCn1) was confirmed by restriction digest. A weaker band at 1295 bp, which was most abundant in aorta, was characterized by cloning and sequencing and represented NBCn1, including the mentioned 368-bp fragment, confirming the existence of two splice variants, of which only one was expressed in any significant amount in the intestine (Fig. 1).


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Fig. 1.   Method of the semiquantitative RT-PCR (A), NBC1 (B), and NBCn1 (C) expression analysis in mouse intestinal epithelia (epith.) and aorta or kidney, respectively. A: The figure shows an example of an amplification of NBC1 from murine colonic crypts. The amplification efficiency of both PCR products, gene of interest and 18s rRNA as control, was determined by calculating the slope following semilogarithmic plotting of the values against the cycle number (line graph). The virtual relationship optical density integrated (ODI) of the studied gene vs. ODI of 18s rRNA was calculated and represents the expression level of the gene of interest after correction of the values for the different PCR products according to their length. However, it is important to realize that a comparison of the expression level of one gene of interest with the expression level of another gene of interest, for example NBC1 with NBCn1, is only possible in an approximate fashion. B: NBC1 expression was examined in mouse intestine. Relative expression levels compared with 18s rRNA are shown in the duodenum (duod.), ileum, colon, and kidney (n = 4-6). C: 2 NBCn1 cDNA fragments were amplified, cloned, and identified as splice variants of NBCn1 by sequence analysis. PCR revealed 890- and 1295-bp bands. The upper band was most abundant in aorta and not significantly expressed in the intestine, whereas the 890-bp band was detected in duodenal as well as colonic mucosa (n = 4-6).

Semiquantitative RT-PCR. Homologous primers for murine NBC1 and NBCn1 were deduced from obtained (NBCn1, see Cloning of murine NBCn1 cDNA fragments) or published sequence information (NBC1: forward primer 5'-TAAACCAGAGAAGGACCAGC-3', reverse primer 5'-CGTCAGACATCAAGGTGGC-3'). An 18s rRNA fragment was amplified as an internal control with the use of primers supplied by Ambion (Austin, TX). Semiquantitative RT-PCR was performed as described previously (3, 30), only using different annealing temperatures (52°C for NBC1, 58°C for NBCn1, and 58°C for 18s rRNA). The relationship of the studied gene vs. 18s rRNA was calculated and regarded as the relative expression level.

pHi measurements. Fluorescence microscopy for determination of pHi was performed as described (5, 30), similar to previously applied techniques (1, 20). After crypts were loaded for 30 min at room temperature with 5 µM BCECF in buffer A, crypts were fixed between a glass coverslip and a 0.3-µm polycarbonate membrane (Osmonics, Minnetonka, MN) on the stage of an inverted microscope (Nikon Diaphot TMD, Nikon, Düsseldorf, Germany) and superfused following the appropriate protocol with buffer A, buffer B (40 mM NaCl replaced by NH4Cl), and buffer C [containing 120 mM tetramethyl ammonium chloride (TMA) instead of NaCl]. For CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solutions, 20 mM NaCl was replaced by Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and buffers were gassed with 95% O2-5% CO2. Crypts were alternately excited at 440 ± 10 and 490 ± 10 nm at a rate of 100/s. Data acquisition and processing were performed using the software provided by the manufacturer (Photon Technology International, Lawrenceville, NJ). Emitted light from the lower portion of the individual crypt (crypt base) was collected through a 510-nm dicroic mirror, a 530-nm long-pass filter, and an adjustable diaphragm and recorded by a photomultiplier. At the end of each experiment, the 440-to-490-nm ratio was calibrated to pHi using the high K+-nigericin method (in mM: 100 K+ gluconate, 40 KCl, 7 HEPES, 1.2 Ca2+ gluconate, 1.2 MgSO4, 20 glucose, 10 µM nigericin, pH 7.4 or 6.6). Background fluorescence was found to be negligible and was not corrected for.

Determination of the intrinsic intracellular buffering power. Determination of the intracellular buffering power (beta i) was performed as described by Boyarsky et al. (8) using increasing or decreasing concentrations of NH4Cl (0.5-80 mM TMA Cl replaced by NH4Cl in buffer C) and measuring the resultant pHi changes. beta i was calculated over a wide pHi range (pH 6.6-7.8), resulting in buffering curves that were not different between CFTR +/+ and CFTR -/- crypts (Fig. 2).


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Fig. 2.   The intrinsic buffering capacity (beta i) was determined in murine colonic crypts between pHi 6.6 and 7.8 as described in the MATERIALS AND METHODS section. The recorded buffering curve with a beta i of 45.1 mM/pH at pHi 6.7, 24.7 mM/pH at pHi 7.1, and 26.7 mM/pH at pHi 7.6 resembles the one in other cell types and was not different between CFTR +/+ and CFTR -/- crypts.

Statistics and calculation of proton fluxes. Results are given as means ± SE. Proton fluxes were calculated by multiplying the initial steep pHi slope after readdition of sodium or removal of NH4Cl, respectively, which was determined by regression analysis, with the total buffering capacity at the initial pHi, (5) including the intrinsic beta i and, in addition, the CO2-dependent buffering capacity for CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions. Statistical significance was determined using Student's t-test.


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In situ hybridization of NBC1 in mouse proximal colon. We had previously observed rather high NBC1 expression levels in rabbit proximal colon (18) and wondered whether colonic crypts, in which the assessment of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport transport rates is feasible using fluorometric techniques to monitor changes in pHi, have substantial NBC1 expression. In situ hybridization revealed that NBC1 expression was indeed primarily located in the cryptal region (Fig. 3).


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Fig. 3.   NBC1 expression along the crypt-villus-axis in murine proximal colon. Adjacent frozen sections were hybridized to sense and antisense NBC probes according to MATERIALS AND METHODS. NBC is detected at high levels only in crypt epithelium. A: hematoxylin and eosin stained section; B: section labeled with antisense probe; C: section labeled with sense probe. Pictures were taken under Nomarski optics. Length of the scale bar is 100 µm.

NBC1 and NBCn1 expression levels in the murine intestine. To have an estimate of the relative Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity that was mediated by NBC1 and NBCn1, another NBC isoform whose expression has recently been reported in the murine duodenum (24), we measured the expression levels of NBC1 and NBCn1 throughout the murine intestine using a semiquantitative PCR technique. Primers were chosen that displayed a similar amplification rate as the primers for 18s rRNA, which was used as an internal control (3). Figure 1 displays the relative expression levels for NBC1 and the two splice variants of NBCn1 in the intestine, kidney, and aorta, respectively. The chosen primers for NBC1 amplify both subtypes, kNBC1 and pNBC1, but we know from previous work that although both isoforms are expressed in the intestine, the pNBC1 has much higher expression levels (32). The expression of only one of the two NBCn1 splice variants that we had cloned was found in the intestine, and expression levels were ~30% of those of NBC1 (Fig. 1). However, we found a strong enrichment of NBC1 in colonic crypts, and we observed the same for the Na+-K+-2 Cl- cotransporter NKCC1 (not shown), whereas the expression of NBCn1 in the whole proximal colon was higher than in crypts, suggesting that this isoform is predominantly expressed in subepithelial layers, possibly muscular tissue.

Assessment of Na+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport activity in murine colonic crypts. Steady-state pH was 7.53 ± 0.02 in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 7.35 ± 0.03 in its presence. In CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, forskolin did not result in a significant change in steady-state pHi. To assess Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in colonic crypts, pHi recovery from an acid load using the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> prepulse technique was measured in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 4A) and in its presence (Fig. 4B), with and without the Na+/H+ exchange (NHE) inhibitor dimethyl-amiloride (DMA). After superfusion with buffer B and buffer C for 5 min each, crypts acidified to pH 6.63 ± 0.03 in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and to pH 6.61 ± 0.02 in its presence and subsequent readdition of Na+ caused a flux rate of 18.73 ± 1.73 and 23.8 ± 1.7 mM/min, respectively. The concentration of 700 µM DMA blocks all membrane-resident Na+/H+ exchanger isoforms expressed in the colon, namely NHE1, NHE2, and NHE3. DMA (700 µM) abolished pHi recovery in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, a DMA-insensitive flux rate of 5.07 ± 0.5 mM/min was observed (Fig. 4, C and D). S0859, an inhibitor of NBC1 Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters [not inhibitory for all isoforms of the NBC gene family, but important for this study, without effect on NHE and anion exchange (AE) (A. Heinzmann, unpublished observations)] reduced the acid extrusion rate to the one found in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions. This finding further substantiates the fact that the DMA-insensitive base influx rate is attributable to Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity. It also suggests that 700 µM DMA, which is a very high concentration but, unfortunately, essential in this study, does at least not strongly inhibit NBC1, because in that instance, we should see a larger S0859-inhibitable than DMA-insensitive acid extrusion rate.


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Fig. 4.   NBC activity in isolated colonic crypts, determined as the acid-activated, dimethyl amiloride (DMA)-insensitive, S0859-sensitive HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent proton/base flux rate (Fig. 4D). A and C: experiments where cells recover from an acid load in HEPES/Tris/O2 (A) and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (C)-buffered medium in the presence and absence of the Na+/H+ exchange inhibitor DMA. See text for explanation of results. F, forskolin (n = 6-8 crypts from 5-6 mice). * P < 0.05; ** P < 0.01.

To assess whether cAMP-dependent stimulation of NBC activity occurs, the same experiments were performed shortly after the addition of forskolin to the perfusate. Forskolin (10 µM) 5 min before acidification increased the DMA-insensitive acid extrusion rates by 62% (Fig. 4D).

Effect of forskolin of Na+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport activity in CFTR +/+ and CFTR -/- mice. One possible mechanism for an indirect activation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport by forskolin could be a secretion-associated cellular shrinkage. CFTR -/- mice do not show forskolin-induced shrinkage (6, 37) and were therefore used to address this question.

CFTR -/- crypts acidified to pH 6.48 ± 0.03 and the Na+-dependent-, DMA-insensitive acid extrusion rate was 3.63 ± 0.17 mM/min, which was 28% less than in crypts of their CFTR +/+ littermates. However, forskolin nevertheless increased NBC activity by 53.3% to 5.56 ± 0.17 mM/min (Fig. 5), which is almost identical to the activation in CFTR +/+ crypts. To test whether the decrease in Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity could be due to reduced NBC1 expression, we determined NBC1 and NBCn1 expression levels in colonic crypts of CFTR -/- mice and their normal littermates. NBC1 expression was not significantly reduced in CFTR -/- crypts.


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Fig. 5.   A: NBC activity in colonic crypts from CFTR +/+ mice and from CFTR -/- mice with and without forskolin stimulation. In CFTR +/+ crypts, forskolin causes a 62% activation of the acid extrusion rate (5.07 ± 0.07-8.20 ± 0.37 mM/min; P < 0.01). However, in the absence of conductive Cl- exit, i.e., in CFTR -/- mice, the flux rate was still enhanced by 61% (3.63 ± 0.17-5.56 ± 0.17 mM/min; P < 0.01). This indicates that CFTR is not necessary for cAMP-induced NBC activation in murine colonic crypts (n = 7-9 crypts from 5-6 mice in each group; ** P < 0.01; n.s., not significant). B and C: relative expression levels of NBC1 (B) and NBCn1 (C) in colonic crypts of CFTR +/+ (filled bars) and CFTR -/- (open bars) mice compared with 18s rRNA. Expression of NBC1 was slightly but not significantly lower in cystic fibrosis colonic crypts. NBCn1 expression levels were very low compared with NBC1 (n = 5 mice in each group).

Effect of K+-channel inhibition on Na+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport activity. Although the expression level of NBC1 in the murine colon far exceeded that of NBCn1, we cannot rule out a colonic expression of additional, not-yet-cloned NBC isoforms. Therefore, we wanted to get some insight into the electrogenicity of colonic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. We speculated that the forskolin-induced activation of chromanol-sensitive K+ channels, which occurs and is obligatory for colonic anion secretion (7), should decrease Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> influx if the coupling of Na+ to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is 1:2 or higher, whereas it would not be influenced if it was 1:1 or dropped to 1:1 on stimulation. We therefore measured the effect of forskolin on the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rate in the presence of 100 µM of the chromanol derivative HOE-293b, which is fully inhibitory for the colonic cAMP-activatable KCNQ1 K+ channels (7). Surprisingly, we found that HOE-293b inhibited acid-activated NBC activity. This may be related to a slight swelling of the crypts after the application of HOE-293b (by ~2%, O. Bachmann, unpublished observations) or to some unknown effect. Forskolin, however, caused a markedly stronger stimulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rate in the presence compared with the absence of HOE-293b (270% vs. 160%, Fig. 6). This shows that attenuating the forskolin-induced hyperpolarization of the basolateral membrane does indeed increase the stimulatory effect of forskolin on Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> activity and suggests that at least a major part of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is membrane potential dependent and, therefore, likely electrogenic. It also supports the conclusions from the previous section, i.e., the independence of the forskolin-induced stimulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity on its stimulation of anion secretion.


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Fig. 6.   Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity during inhibition of chromanol-sensitive K+ channels. HOE-293b inhibited the basal cotransport rate. However, forskolin caused a stronger stimulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in the presence of 293b than in its absence (n = 8-9 crypts from 5-6 mice in each group; ** P < 0.01).


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ABSTRACT
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This study investigates colonic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter expression and localization and addresses the question whether an increase in intracellular cAMP stimulates colonic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity and whether this stimulation is secondary to cAMP-induced apical anion secretion or a cAMP-induced change in cotransport activity itself.

Apparent Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity has been found in multiple tissues including the GI tract (10, 14, 26, 36, 40). Data from frog stomach (10) suggested an inward direction of Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the resting state, in contrast to that in the proximal tubule, and therefore a coupling of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> of 1:2 or less. Later, evidence for the involvement of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters in pancreatic and duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (11, 17) and even more recently in proximal colonic Cl- secretion has been obtained (19), suggesting an inward flux of Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> even in the stimulated state in these epithelia. Also, evidence was obtained for a direct or indirect stimulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity during agonist-stimulated secretion (18).

The first cloning of an Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter cDNA from salamander kidney, now named kNBC1, in 1997 (29) allowed the rapid detection of members of this HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter gene family in a variety of organs. Of the cloned members of the NBC family, we found the highest expression for the NBC1 isoform throughout the rabbit intestinal tract (34). We also observed that the NBC1 mRNA in stomach, pancreas, and all parts of the intestine was 1.8 kb larger than that of rabbit kidney cortex, suggesting different molecular entities in GI tract and kidney (18). When we cloned the complete rabbit intestinal NBC1 and the NH2 terminus of rabbit kidney cortex NBC1 by rapid amplification of cDNA ends (RACE)-PCR, we found that the kidney and intestinal NBC1 differed in the cytoplasmic NH2 terminus only. RT-PCR with primers that distinguished between the two subtypes revealed that both subtypes were expressed in the intestine but with a very strong quantitative predominance of the intestinal subtype (32). A similar strictly NH2-terminal sequence variation was observed for NBC1 from human kidney cortex and pancreas and the two isoforms called kNBC1 and pNBC1 (2).

These findings raised two interesting questions. First, is the intestinal/pancreatic NBC1 stimulated by a rise in intracellular cAMP, as suggested by indirect evidence? Second, could it be that the difference in the NH2 terminus between the two NBC1 subtypes, far away from the actual membrane-spanning part of the molecule, was the reason for the presumably different coupling ratios between Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the proximal tubule and the intestine?

Functional data demonstrate an inhibition of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity during a rise in intracellular cAMP in the proximal tubule. Because the Na+ uptake pathway in the apical membrane is the Na+/H+ exchanger isoform NHE3, whose transport activity is inhibited by PKA-dependent phosphorylation (21, 39, 41), the effect of a rise in cAMP on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity could be secondary to the inhibition of apical NHE. In the intestine and pancreas, a rise in intracellular cAMP stimulates apical Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and indirect evidence suggests a concomitant stimulation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity (16, 18). Again, the effect of cAMP on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> contransport could be secondary to agonist-stimulated anion secretion.

We, therefore, searched for a model in which we could investigate whether an increase in intracellular cAMP stimulates the intestinal/pancreatic NBC1 subtype in a native cellular system and whether this stimulation was secondary to cAMP-stimulated secretion or a direct effect on the cotransporter(s). Isolated colonic crypts are a model in which vectorial anion secretion is still intact, as demonstrated by marked cAMP-induced shrinkage and loss of intracellular Cl- after forskolin stimulation (6). They also allow direct measurement of acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity by microfluorometry (30). In addition, they can be prepared from CFTR -/- mice, which exhibit no electrogenic anion secretory response in the colon. NBC1 in situ hybridization demonstrated that NBC1 expression was located almost exclusively in the cryptal region, which made isolated colonic crypts an ideal model to study the above questions. Colonic NBCn1 expression levels were approximately one-third those for NBC1 in total colonic tissue, but although we observed much higher NBC1 expression levels in colonic crypts than total colon, NBCn1 levels were markedly lower in crypts than total colon. This indicates that NBCn1 is most likely predominantly nonepithelial in the colon. We know from previous experiments that expression of NBC3 (Cl--dependent NBC) is very low throughout the intestinal tract (18) and that although some kNBC1 is expressed in the colon, the pNBC1 subtype is expressed at far higher levels (32).

Forskolin increased the acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rate by 62%. The acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport rate likely reflects maximal or near-maximal transport rate because low pH stimulates Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> activity and because the ion gradients are optimized for maximal driving force. Thus it is per se extremely unlikely that a change in intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration {[HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i} could explain the forskolin-induced increase in transport rate, because we already pose a large, but equal, change in [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i on the cells. In addition, we did not observe such a change, even in the absence of acid activation. This may have something to do with the fact that in the colon, the major role of Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in anion secretion is the uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in exchange for Cl- via a basolateral AE (19). Associated pHi shifts, if they occur at all, may be so local that single-cell microfluorometry does not pick them up.

However, colonic crypts may shrink during agonist-activated secretion, and it is feasible that this shrinkage, rather than the increase in cAMP itself, increases Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity. We therefore investigated the effect of forskolin on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in crypts isolated from CFTR-deficient mice. These mice show no increase in short-circuit current (Isc) on stimulation with cAMP analogs, and we have found that in contrast to crypts isolated from normal littermates, they do not shrink on forskolin stimulation (6). When acidified to the same low pHi, Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity was ~30% lower than that in crypts from normal littermates. The somewhat lower Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rates are accompanied by slightly lower NBC1 expression levels in CFTR -/- crypts. Interestingly, activation by forskolin was by 54% and, therefore, similar than in normal littermates. These finding demonstrate that Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> activation by forskolin is not dependent on apical anion secretion.

Thus, in the absence of CFTR expression, Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity is indeed suppressed in native colonic crypts, as recently reported by Shumaker et al. (35) in the pancreatic cell line CF-PAC. However, the difference in NBC expression and activity, between normal and CFTR-deficient native crypts, was much smaller than that between CFTR-deficient and CFTR-retransfected CF-PAC cells, and activation by cAMP was normal in crypts. One possible explanation for the discrepancy in the results obtained in CF-PAC cells and native colonic crypts is that the rapidly growing tumor cell line CF-PAC has lost much of its ability to perform vectorial ion transport, and that retransfection of CFTR slows its growth rate allowing for differentiation into a transporting epithelium.

We next performed experiments with the chromanol HOE-293b. This substance inhibits a certain type of K+ channels, and Bleich and Warth (7) recently demonstrated that the inhibition of these KCNQ channels virtually abolishes rat colonic electrogenic anion secretion. We have tested the effect of HOE-293b on the forskolin-induced Isc response in murine proximal colon, and we also found a strong reduction (albeit no abolition) of the secretory response by 100 µM HOE-293b. The rationale of measuring the effect of forskolin on Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rate in the absence and presence of HOE-293b was twofold. First, this treatment is an alternative mode of inhibiting apical anion secretion; thus we could validate our findings in the CFTR -/- crypts. Second, and more importantly, the prevention of forskolin-induced hyperpolarization of the basolateral membrane potential (via KCNQ-channel activation) allows insight into the electrogenicity of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport that we studied. We found that the prevention of forskolin-induced hyperpolarization greatly augments the stimulatory effect of a rise in intracellular cAMP on Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> activity, suggesting electrogenicity of at least a major part of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport.

Gross et al. (12, 13) found a coupling ratio for Na+ to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> of 1:2 in pancreatic cells and the same ratio when kNBC1 was expressed in an intestinal cell line, whereas it was 1:3 in a renal cell line, suggesting that it is the cell type, not the NBC1 sequence, that determines the coupling ratio. Müller-Berger et al. (23) presented evidence suggesting that the Ca2+-dependent stimulation status of the cell determined the coupling ratio of NBC both in native proximal tubules and in kNBC1-expressing oocytes, and recently, Gross et al. (13) presented evidence for a decrease in the coupling ratio between Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by PKA-mediated phosphorylation in heterologously expressed kNBC1.

It would be highly interesting to study the stoichiometriy of the pNBC1 and a potential stimulation-associated change. Unfortunately, our experimental model is not suitable to definitely determine the coupling ratio for Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in colonic crypt Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. Given the fact that obviously different NBC isoforms and subtypes are expressed in the colon, the attempt to do so is of questionable value.

In summary, this study demonstrates that the intestinal/pancreatic subtype of NBC1 is strongly expressed in the colon and located predominantly in the crypt region. NBCn1 is also expressed, with far lower expression levels. A rise in intracellular cAMP increases acid-activated Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity, independently of secondary changes in cell volume or anion secretory activity. Preventing the forskolin-induced hyperpolarization strongly increases the stimulatory effect of forskolin, suggesting that at least a major part of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in colonic crypts is electrogenic.


    ACKNOWLEDGEMENTS

We thank C. Neff for expert technical assistance; Drs. A. Weichert and W. Kleemann from Aventis Pharma, Frankfurt, for kindly providing the S0859 compound; Prof. D. Mecke for the use of the animal facilities in the Dept. of Biochemistry of Tübingen University; and Prof. Dr. Michael Sessler, Dept. of Surgery of the Univ. of Tübingen, for constructive criticism.


    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Grants Se-460/13-1 and Se-460/13-2, by the Mukoviszidose e.V., and by a grant from the Interdisziplinäres Zentrum für Klinische Forschung Tübingen (Project IIIC1).

Address for reprint requests and other correspondence: U. Seidler, Dept. of Gastroenterology and Hepatology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623 Hannover, 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.

September 18, 2002;10.1152/ajpgi.00209.2002

Received 3 June 2002; accepted in final form 10 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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