Journal of the Pancreas Open Access

Keywords

Anions; Bicarbonates; Colon; Cystic Fibrosis; Duodenum; Intestinal Mucosa; Intestinal Obstruction; Intestinal Secretions; Ion Transport

Abbreviations

AE: anion exchanger; BCECF: 2’7’-bis(2-carboxyethyl)-5(6)- carboxyfluorescein; BLMvs: basolateral membrane vesicles; [Cl–]i; intracellular Clconcentration; CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator; DIDS: 4,4'-diisothiocyanatostilbene-2,2'- disulphonic acid; DMA: dimethyl-amiloride; dNBC1: HCO3– cotransporter isoform I; GAPDH: glycerol aldehyde trisphosphate dehydrogenase; Hoe: Hoechst compound; Isc: short-circuit current; kNBC1: kidney NBC1 subtype; NBC: Na+HCO3– cotransport; NHE: Na+/H+ exchanger; NKCC: Na+K+2Clcotransport; pHi: intracellular pH; pNBC1: pancreatic NBC1 subtype; RACE PCR: rapid amplification of cDNA ends PCR

In cystic fibrosis (CF) patients and CFTR (-/-) mice, intestinal, pancreatic and biliary HCO3– secretion is impaired. In all segments of in vitro CFTR (-/-) mouse intestine, we found a reduction in basal HCO3– secretory rate and a striking absence of a HCO3– secretory response to all tested physiologic agonists [1].

Since small intestinal peptic damage, malabsorption, and obstruction of biliary and pancreatic ducts as well as the intestinal lumen in CF patients may, at least in part, be secondary to the HCO3– secretory defect, we further investigated intestinal HCO3– transport pathways in normal and CF epithelium. The curious observation was that despite the molecular or functional presence of non-CFTR anion channels in the intestine of CFTR (-/-) mice, and despite the fact that HCO3– secretion could be stimulated in CFTR (-/-) intestine using tools to alter enterocyte membrane potential, no physiologic stimulus was found that caused significant HCO3– secretion.

Recent investigations have revealed a plethora of secondary defects in cells not expressing the CFTR protein, including defective expression and/or regulation of ion transport proteins [2-8]. We therefore speculated that downregulation of expression or defective regulation of basolateral HCO3– uptake mechanisms may intensify the HCO3– secretory defect in CF intestine and possibly have secondary effects on the intracellular pH (pHi) and volume regulatory or absorptive function of CF enterocytes. The HCO3– supply pathways during intestinal HCO3– secretion were ill defined, but some functional evidence existed that a stilbenesensitive transport pathway, possibly a Na+HCO3– cotransporter, was involved in HCO3– secretion in the duodenum, the intestinal segment with the highest HCO3– secretory rate [9, 10, 11].

We therefore aimed to 1) functionally identify the major base uptake mechanisms in isolated duodenal basolateral membranes, 2) clone and sequence potential intestinal base uptake mechanisms and demonstrate their physiological function in the HCO3– secretory process and 3) study their expression levels and regulation in normal and CF intestine.

Na+HCO3– Cotransport in the Duodenum

Functional identification of the major base uptake mechanisms in duodenal basolateral membrane vesicles. Previous experiments had demonstrated that duodenal HCO3– secretion [9, 11] and base uptake into isolated duodenal cells [12, 13, 14] is Na+-dependent. Thus, the investigation of pHi-gradient driven Na+ uptake should assess all physiological base uptake mechanisms in the duodenocyte. Figure 1A shows 22Na+ uptake into rabbit duodenal basolateral membrane vesicles (BLMvs) in the absence and presence of HCO3–, with and without dimethyl-amiloride (DMA), an inhibitor of Na+/H+ exchange, and in the absence and presence of a pH-gradient. It is obvious that pHi-dependent 22Na+ uptake consists of a HCO3–-dependent, DMA insensitive one (Na+HCO3– cotransport) and a DMA-sensitive, HCO3– independent one (Na+/H+ exchange). Consistent with this concept was the finding that 4,4'- diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) inhibited the HCO3– dependent, DMA insensitive, pH-gradient driven 22Na+ uptake, whereas more than 80% of DMA-sensitive, HCO3– -independent 22Na+ uptake was inhibited by 1 μM Hoe 642 (Hoechst compound 642) and therefore mediated by the NHE1 isoform of the Na+/H+ exchanger (NHE) gene family (data shown in [15]).

Molecular characterization and expression levels of duodenal basolateral HCO3– transporters. Having obtained functional evidence for the presence of a Na+HCO3– cotransporter in the basolateral membrane of rabbit duodenocytes, we wanted to establish its molecular identity. We therefore cloned and sequenced cDNA fragments for all recently cloned NBC isoforms, and established their expression levels by a semiquantitative PCR protocol (data shown in [15]). Although other isoforms like NBC2 [16] and NBCn1 [17] were expressed in the intestinal tract, the NBC1 isoform had the highest expression levels. Figure 1B shows a Northern Blot with poly (A+) RNA from different gastrointestinal segments and kidney cortex, hybridized with cDNA fragments for NBC1 and NHE1, the two major basolateral base uptake mechanisms in rabbit duodenocyte. Both NBC1 and NHE1 are strongly expressed in duodenal mucosa. Since we observed a difference in mRNA size between NBC1 expressed in the intestine and the kidney, we cloned the complete NBC1 sequence from rabbit duodenum and pancreas and the N-terminal sequence from rabbit kidney and found that the gastrointestinal and the kidney isoform differ in their N-termini. Primers were chosen for the selective amplification of the different N-termini from the different gastrointestinal segments and kidney cortex. Figure 2 demonstrates that both NBC1 subtypes are expressed in intestine and kidney, but with a strong predominance of the intestinal NBC1 (dNBC1) subtype in the intestine and the kidney NBC1 (kNBC1) subtype in the kidney.

Thus, it is clear that Na+HCO3– cotransport in the intestine is mediated by different molecular entities, among which the intestinal subtype of NBC1 has the highest expression levels.

Physiological role of Na+HCO3– cotransport and NHE1 in HCO3– secretion. After having identified a Na+HCO3– cotransporter and NHE1 as the major basolateral base importers in rabbit duodenocyte basolateral membrane, we studied their importance for basal and stimulated duodenal HCO3– secretion. 1 mM DIDS, which in this concentration will inhibit all currently known Na+HCO3– cotransporters, caused a strong reduction of basal ouabain-sensitive HCO3– secretion to about 50%, demonstrating that a major part of the actively secreted HCO3– ions are supplied by uptake via Na+HCO3– cotransport. Unexpectedly, the 8-Br-cAMPstimulated increase in HCO3– secretion was unchanged from the control conditions (Figure 3AB). This suggested that another system for HCO3– supply was activated during secretion. Since the removal of CO2/HCO3– diminished basal and stimulated HCO3– secretion by approximately 85% (data shown in [15]), only 15% can be generated by CO2 hydration from intracellular sources or by import and secretion of a base other than HCO3–. Thus, the likely alternative system was CO2 uptake and hydration via carboanhydrase, and basolateral extrusion of the generated protons by Na+/H+ exchange. 1 mM acetazolamide caused ouabain-sensitive basal secretory rate to drop to approximately 50% of control value, but surprisingly, the 8-Br-cAMP-stimulated increase in HCO3– secretion was not diminished compared to the control, suggesting upregulation of Na+HCO3– cotransport (Figure 3C). Only the combination of DIDS and acetazolamide, or DIDS and 1 μM Hoe 642, which will inhibit NHE1 and thereby the basolateral extrusion of protons generated during CO2 hydration, strongly reduced both the peak and the duration of the secretory response (Figure 3DE). These results demonstrate that Na+HCO3– cotransport and CO2 hydration/Na+/H+ exchange via NHE1 are equally important pathways for duodenal HCO3– supply and are upregulated during cAMP-mediated stimulation

Na+HCO3– Cotransport in the Colon

The Northern Blot in Figure 1 shows a strong NBC1 expression in the proximal colon as well as the duodenum. However, HCO3– secretion is low in this part of the intestinal tract. We therefore wondered what might be the physiological significance of this transporter in the colon. HCO3– ions are exchanged for luminal Cl– in the process of electroneutral salt absorption, and HCO3– ions for this process could be in part taken up by Na+HCO3– cotransport. However, this would imply the concomitant uptake of Na+ ions from the interstitium, a process that would seem somewhat counterproductive for NaCl absorption. On the other hand, Na+HCO3– cotransport, coupled to an electroneutral Cl– /HCO3– exchange mechanism in the basolateral membrane, could serve as an alternative Cluptake mechanism to the Na+K+2Clcotransporter during Cl– secretion. This concept would be consistent with earlier observations of residual anion secretion after pharmacological or gene technologic inhibition of Na+K+2Clcotransport [18, 19], as well as explain the high colonic expression levels for the basolateral anion exchanger AE2 [20]. To test this hypothesis, we measured 8-Br-cAMPstimulated short-circuit current (Isc) and HCO3– secretion in murine muscle-stripped proximal colon in the Ussing-chamber. Serosal bumetanide (100 μM) or the NBC-specific inhibitor S0859 (100 μM) reduced db-cAMPinduced DIsc by 60% and 45%, respectively, the combination of both plus bilateral acetazo lamide inhibited DIsc to the same extent as bumetanide in the absence of CO2/HCO3– (Figure 4A-D). Acetazolamide augmented the inhibitory effect of S0859 but not of serosal DIDS (inhibits NBC and AE2), suggesting that the HCO3– ions used for basolateral Cl–/HCO3– exchange (via AE2) are predominantly supplied by basolateral Na+HCO3– cotransport and to a minor extent by CO2 hydration (data not shown). These results demonstrate that in the colon, basolateral Na+HCO3– cotransport is involved in Cl– uptake during colonic anion secretion.

NBC1 and NKCC1 Expression and Regulation in Normal and CF Colonic Crypts

Na+HCO3– and Na+K+2Cl– cotransport is activated during cAMP-stimulated anion secretion, but whether this is a direct effect of the second messenger or secondary to cell volume reduction subsequent to Cl– channel opening (which by itself can activate protein kinases [21, 22, 23]), or both, is under debate [24-30]. We wondered if reduced expression levels or defective regulation of these anion uptake pathways may be a component of the secretory defect in CF epithelia. We did indeed observe a reduction in NBC1 and NKCC1 expression in the intestine of adult CFTR (-/-) mice in relation to the 18s rRNA, but not when compared to villin, a cytosceletal protein of a brush border membrane, suggesting that structural changes may occur in the intestine of these mice (Figure 5). Therefore, we searched for a model in which the activity of Na+HCO3– and Na+K+2Cl– cotransport could be assessed at a cellular level. Colonic cypts were isolated from the proximal colon of CFTR (-/-) mice and their normal littermates and NBC and NKCC activity were assessed fluorometrically after loading the crypts with the pHi-sensitive dye 2’7’-bis(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF). Crypt volume was measured by determining the cross sectional area of calcein-loaded crypts using confocal microscopy. We found that although basal NBC and NKCC activity were slightly reduced in CF crypts, forskolin activated both NBC and NKCC transport activity to the same degree in CFTR (+/+) and (-/-) crypts (data not shown). Forskolin stimulation caused a marked reduction in the crypt cross sectional area in CFTR (+/+) crypts, whereas no change in crypt volume was seen in CFTR (-/-) cells. This demonstrates that changes in cell volume is not the mechanism by which forskolin activates NKCC and NBC transport activity.

In summary, we found that the intestinal subtype of the NBC1 is strongly expressed in both the duodenum and colon. In the duodenum, its expression is in the villus region and it is a major HCO3– supply pathway during basal and stimulated HCO3– secretion. In the colon, it is expressed predominantly in the crypts and one of its physiological functions is to serve as an alternative Cl– uptake mechanism in conjunction with a basolateral anion exchanger. Our current concept of the anion transport mechanisms in duodenum and colon is depicted in Figure 6.

Methods

BLMV isolation. Basolateral membrane vesicles from rabbit duodenum were isolated by a combination of differential and sucrose density gradient centrifugation as previously described [15, 31].

22Na+ uptake experiments. To load BLMvs with the appropriate intravesiclar buffer, BLMvs were suspended, centrifuged and revesiculated in the appropriate buffer. To avoid the buildup of a diffusion potential, 20 μM valinomycin and high and equal K+ concentrations were present in the intravesiclar and uptake buffer. Details of the method and the appropriate buffer compositions are described elsewhere [15].

Cloning and expression studies To clone rabbit NBC1, degenerate primers were chosen based on the published sequence from ambystoma and human kidney [32, 33]. The N-termini of the intestinal and kidney NBC1 subtype were cloned by rapid amplification of cDNA ends PCR (RACE PCR). Based on established cDNA sequences, appropriate primers for quantitative PCR analysis were searched for and tested, and a semiquantitative PCR protocol was established in which the amplification of the gene of interested was compared to that of a control gene in the exponential phase of the reaction [34].

Ussing-chamber experiments Muscle-stripped intestinal segments were placed in conventional Ussing-chambers, electrical parameters were recorded during continuous or intermittent voltage-clamp, and HCO3– secretion was measured by pH-stat titration.

Colonic surface cell and crypt isolation Colon crypts were isolated by vibration of everted proximal colon in EDTA-containing solutions, and sequential isolation of surface cells, then a few fractions that were discarded, and eventially whole crypts.

Fluorescence measurements To measure NBC transport rates, BCECF-loaded cypts were acidloaded by an ammonium prepulse, and pHirecovery measured in the presence of 700 μM dimethyl-amiloride (DMA), which blocks all sodium-proton-exchanger isoforms in the colon. The corresponding flux rates were calculated from the pHi recovery rate and the buffering capacity at the appropriate pH. To measure NKCC activity, we utilized the fact that NH4 + can enter the cell via the NKCC, using the K+ transport site, and thereby causes a proton influx. After superfusion of the crypts with ammonium chloride, pHi first increases sharply, because the cell permeable NH3 causes an alkalinization. The pHi recovery that follows is in part mediated by NH4 uptake by NKCC. The base flux rate after ammonium pulse is mainly composed of an azosemide fraction, representing NKCC activity, and a DIDSsensitive fraction which reflects the activation of a Cl–/base exchanger.

Crypt volume measurements Confocal images of the crypt cross sectional area were used as a measure for the crypt volume as described by Heitzmann et al. [27].

Acknowledgements

The authors thank Dorothee-Vieillard-Baron and Christina Neff for excellent technical help, William Colledge, Rosemary Ratcliffe and Michael Evans for making a CFTR +/- breeder pairs available, Jens Leipziger for help with the volume measurements, Detlef Wermelskirchen and Barbara Osikowska for help with the [Cl–]i measurements, Dieter Mecke for the use of the animal facility and Richard Wahl for use of the isotope laboratory. We thank Walter Boron, Urs Berger, Bernhard Schmitt, Robert Müller, Gary Shull, Seth Alper, Juha Kere, Jon Isenberg and Gunnar Flemström for helpful discussions.

The work was supported by the DFG grants Se 460/9-1 – 9-4, Se 460/13-1 – 13-2, by a grant from the Federal Ministry of Education, Science, Research and Technology (Fö 01KS9602) and the IZKF Tübingen, and by a grant from the Deutsche Mukoviszidosestiftung e.V.

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

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