Intestinal ion transport in NKCC1-deficient mice

B. R. Grubb, E. Lee, A. J. Pace, B. H. Koller, and R. C. Boucher

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The Na+-K+-2Cl- cotransporter (NKCC1) located on the basolateral membrane of intestinal epithelia has been postulated to be the major basolateral Cl- entry pathway. With targeted mutagenesis, mice deficient in the NKCC1 protein were generated. The basal short-circuit current did not differ between normal and NKCC1 -/- jejuna. In the -/- jejuna, the forskolin response (22 µA/cm2; bumetanide insensitive) was significantly attenuated compared with the bumetanide-sensitive response (52 µA/cm2) in normal tissue. Ion-replacement studies demonstrated that the forskolin response in the NKCC1 -/- jejuna was HCO3- dependent, whereas in the normal jejuna it was independent of the HCO3- concentration in the buffer. NKCC1 -/- ceca exhibited a forskolin response that did not differ significantly from that of normal ceca, but unlike that of normal ceca, was bumetanide insensitive. Ion-substitution studies suggested that basolateral HCO3- as well as Cl- entry (via non-NKCC1) paths played a role in the NKCC1 -/- secretory response. In contrast to cystic fibrosis mice, which lack both basal and stimulated Cl- secretion and exhibit severe intestinal pathology, the absence of intestinal pathology in NKCC1 -/- mice likely reflects the ability of the intestine to secrete HCO3- and Cl- by basolateral entry mechanisms independent of NKCC1.

sodium-potassium-chlorine ion cotransporter; chloride secretion; bicarbonate secretion; jejunum; cecum


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INTRODUCTION
MATERIALS AND METHODS
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INTESTINAL SECRETION IS IMPORTANT in maintaining the liquidity of intestinal contents during digestion, which aids the mixing of digestive enzymes with the luminal contents. In addition, the liquid secreted in the crypts serves to flush the mucus produced in the crypts to the surface epithelium, where it protects and lubricates the mucosa. In most intestinal regions, Cl- has been thought to be the primary anion secreted, which induces liquid secretion isosmotically. Defective regulation of Cl- secretion underlies major intestinal pathophysiology ranging from diarrhea (hypersecretion of liquid) to cystic fibrosis (CF; hyposecretion of liquid).

Studies in the CF mouse have established that an absence of Cl- secretion is associated with crypt dilation, goblet cell hyperplasia, and intestinal obstruction (see Ref. 8 for review). In light of the Cl- channel functions described for the CF transmembrane conductance regulator (CFTR) and its apical membrane localization in gut epithelia, the data from the CF mouse have established CFTR as the major path for conductive Cl- exit across the enterocyte apical membrane during anion secretion.

Although the focus of much research has been on the apical Cl- channel (CFTR) responsible for intestinal secretion, the basolateral pathway(s) by which Cl- enters the cell has received less attention. The basolateral Na+-K+-2Cl- cotransporter (NKCC1) is thought to play a key role as a Cl- entry pathway in intestinal epithelia (9, 11) to sustain Cl- secretion. In intestinal epithelia, NKCC1 electroneutrally transports Cl- across the basolateral membrane along with Na+ and K+, with a stoichiometry of 1 Na+:1 K+:2 Cl- (9). The loop diuretics bumetanide and furosemide are effective blockers of NKCC1 and have been used to determine the contribution of the cotransporter to the secretory response of the intestine. However, a more specific strategy to identify the contribution of basolateral NKCC1 to the basal and stimulated Cl- secretion of the intestine is the use of gene targeting. In the present study, we have inactivated Slc12a2, the gene that codes for the NKCC1 cotransporter, and tested the intestinal epithelia of the mutant mice for the expression of a gut phenotype in Ussing chamber studies designed to identify the NKCC1 cotransporter contribution to basal and regulated anion secretion in two different regions of the mouse gastrointestinal tract.


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Adult animals (3-8 mo old) were used in this investigation. All mice were allowed food and water ad libitum until the time of the experiment. In all cases, homozygous normal littermates (referred to as +/+, normal, or wild-type) were compared with the NKCC1-deficient mice homozygous for mutant Slc12a2 (referred to as NKCC1 -/-). Two mutations in Slc12a2 were generated (20), and mice bearing these mutations were studied. Ion-transport data for only one of the mutations (Slc12a2Delta 1065-1137) are reported, because the physiological characterization of tissues from mice bearing the two mutations was identical. Mice carrying each mutation (not all mice studied were weighed) had a lower body mass than that of normal littermates [19.7 ± 1.2 and 29.9 ± 1.8 g for Slc12a2Delta 1065-1137 mice vs. normal littermates, respectively (n = 6 for each group); 18.7 ± 0.77 and 24.2 ± 0.72 g for Slc12a2Delta 506-621 mice vs. normal littermates, respectively (n = 5 for each group)]. Mice were killed with CO2, and the intestine was quickly removed and placed in oxygenated Krebs buffer. The designated region of the intestine was opened lengthwise, flushed with buffer to remove intestinal contents, and then mounted on Ussing chambers having an exposed surface area of 0.25 cm2 (7). All preparations studied (ion transport and Northern analysis) were "full thickness" with the muscle layer and the enteric nervous system still intact. Tissues were studied under short-circuit current (Isc) conditions for the duration of the experiment. A constant voltage pulse (1-5 mV, 1-s duration) was applied to the tissue every minute. Potential difference and resistance were calculated using Ohm's law from the changes in Isc in response to the voltage pulse.

Solutions and drugs. Several different solutions were used in this investigation. Unless otherwise indicated, the tissues were mounted and bathed in Krebs-HCO3- Ringer (KBR) solution having the following composition (in mM): 140 Na+, 120 Cl-, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO42-, 0.4 H2PO4-, and 25 HCO3-. A Cl--free buffer was used to determine the contribution of Cl- to the basal and stimulated Isc. For a Cl--free, HCO3--replete buffer, 115 mM Na+ gluconate replaced NaCl, 1.2 mM MgSO4 replaced MgCl2, and 1.2 mM CaCl2 was replaced by calcium gluconate (6 mM calcium gluconate was added to offset the calcium-chelating effects of gluconate). In some preparations, basolateral Na+-free buffer was used. For an Na+-free buffer, the solution was identical to KBR except that N-methyl-D-glucamine replaced all Na+. These three buffers were gassed with 95%O2-5% CO2 to maintain pH 7.4. The Cl--free, HCO3--free buffer was similar to the Cl--free buffer except that 125 mM Na+ gluconate was used to replace all Cl- and HCO3- was replaced by 10 mM HEPES buffer titrated to pH 7.4 with 1 M Tris base. The HCO3--free and Cl--free, HCO3--free buffers were gassed with 100% O2. Whenever HCO3- was removed from the medium, 10-3 M acetazolamide was added to the buffer to prevent endogenous tissue HCO3- production. Where indicated, some preparations were treated with TTX (10-6 M basolateral) to block neurotransmitter release from the enteric nervous system. All preparations contained 5 mM glucose in the basolateral solution and 5 mM mannitol in the apical solution.

Selected drugs were used to characterize the basolateral anion entry paths (see Fig. 9). Bumetanide, an inhibitor of NKCC1, was added to the basolateral bath to test for inhibition of the basal Isc or to test for inhibition of the forskolin-stimulated current. The bumetanide sensitivity was calculated as the magnitude of change in Isc over a 4-min period after bumetanide addition. DIDS was used to block both Cl-/HCO3- exchange and Na+-HCO3- cotransport. Dimethylamiloride (DMA) was used to block Na+/H+ exchange. Hydrochlorothiazide, which blocks NaCl-coupled exchange (see Ref. 9 for review), was also tested in intestinal tissue. Drugs were used at the following concentrations (in M): 10-4 bumetanide (basolateral), 10-5 forskolin (bilateral), 10-3 acetazolamide (bilateral), 10-3 DIDS (basolateral), 10-5 DMA (basolateral), 10-4 hydrochlorothiazide (basolateral), and 10-4 amiloride (apical). All drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO) with the exception of DIDS, which was purchased from Molecular Probes (Eugene, OR).

Northern analysis. Total RNA was isolated from salivary and intestinal tract tissue of wild-type animals. Tissue was frozen, homogenized, and phenol/chloroform extracted using RNAzol B (Tel-test, Friendswood, TX). Total RNA (20 µg) was electrophoresed on a 1.1% formaldehyde, 1.2% agarose denaturing gel in the presence of ethidium as described by Kroczek and Siebert (16) and transferred to Immobilon-NC nitrocellulose membrane (Millipore, Bedford, MA) by capillary transfer. Radiolabeled DNA probes were hybridized to Northern blots in Quick-Hybe (Stratagene) for 1 h at 68°C.

Histological analysis. Wild-type and Slc12a2Delta 506-621 mice were killed, and intestinal tract tissues were fixed in 10% neutral-buffered formalin. The fixed tissues were embedded in paraffin, cut into 5-µm sections, and stained with aniline blue and periodic acid-Schiff's reagent.

In situ analysis. A probe for in situ analysis was prepared in the following manner. The primers NKCC3a, 5'-CAG GGC CTG CTT TACTTCATCTTG-3', and NKCC3b, 5'-GCC TTT CCG TGC GAC TGG-3', were used to generate a 1.2-kb cDNA probe from salivary gland mRNA by RT-PCR. The fragment was cloned into pCR 2.1, the clone was digested with Hind III to remove a 700-bp fragment of cDNA, and this clone was religated to give a cDNA probe of ~600 bp corresponding to bases 3127 to 3636 of the published mouse NKCC1 sequence (18). Using this construct, 35S-labeled sense and antisense RNA was prepared (Maxiscript SP6/T7 kit; Ambion, Austin, TX) and used to analyze tissue sections prepared as follows. Tissues were fixed in 4% paraformaldehyde and washed with 30% sucrose in PBS to remove the fixative. Tissues were embedded in Tissue-Tek, and cryostat sections cut at 8 µm were mounted on slides and stored at -80°C. Sections were fixed in 4% paraformaldehyde and dehydrated in a graded series of ethanol washes. Sections were then digested with proteinase K (10 µg/ml) for 30 min at 30°C. Proteinase K was inactivated by addition of 4% paraformaldehyde, and the sections were rinsed in triethanolamine and acetylated with 0.25% acetic anhydride for 10 min. Sections were then rinsed in 0.2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and dehydrated. A quantity of NKCC1 sense or antisense probe producing 1 × 107 cpm was hydridized to the sections overnight at 54°C in 50% formamide, 1× Denhardt's solution, 0.6 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, 0.1% SDS, 10 mM dithiothreitol (DTT), 1 mg/ml yeast transfer RNA, and 10% dextran sulfate. Slides were washed with 4× SSC and treated with 20 µg/ml RNase at 37°C, then washed four times with 2× SSC and 1 mM DTT at room temperature and washed three times with 0.5× SSC and 1 mM DTT at 54°C. Slides were dehydrated and hand-dipped in NTB2 emulsion (Eastman Kodak, Rochester, NY), exposed for 2 wk at 4°C, developed, and stained with hematoxylin and eosin.

Statistics. All data are means ± SE. Only one preparation per animal (of each intestinal region) was studied for each protocol. Data were compared by a Student's t-test if only two groups were being compared. If more than two groups were compared, ANOVA was used and a Student-Newman-Keuls test was used for multiple comparisons among groups.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Expression of NKCC1 in mouse intestinal tract. To examine the expression of Slc12a2 in the mouse intestinal tract, Northern analyses were carried out on RNA prepared from various regions of the intestinal tract of normal animals. Expression was detected in all regions examined; however, expression was higher in the distal regions, with the highest levels seen in the cecum and colon (Fig. 1A). In situ analysis of a section from wild-type jejunum was performed to determine the expression pattern of the message for the Slc12a2 gene within this tissue. Slc12a2 expression was largely restricted to the epithelium lining the crypts (Fig. 1C). No expression was detected in the muscle layers of the intestinal tract.


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Fig. 1.   A: expression of Slc12a2 [which codes for Na+-K+-2Cl- cotransporter (NKCC1)] in the mouse intestinal tract as shown by Northern blot analysis of RNA prepared from various regions of the intestinal tract and the salivary gland of normal mice. Slc12a2 expression increases in the distal regions of the intestinal tract; however, even in these regions, expression levels are not as high as those observed in the salivary gland. B and C: in situ analysis of Slc12a2 expression in the mouse jejunum indicates that expression is largely confined to the crypts; bars = 200 µm. The section shown in B is hybridized with a probe corresponding to the sense strand of the Slc12a2 transcript, whereas that shown in C is analyzed with antisense probe. D: normal adult mouse jejunum. E: NKCC1 -/- mouse jejunum exhibiting scattered dilated crypts (arrows). D and E: stained with aniline blue and periodic acid-Schiff's reagent; bars = 50 µm.

Neonatal death associated with intestinal obstruction is common in mice deficient in the CFTR gene, and the growth rate of the surviving pups is often lower than that of normal littermates (see Ref. 8 for review). No neonatal deaths were observed in the NKCC1 -/- mice in this study, in contrast with data previously described (5). Mice deficient in NKCC1 were growth retarded, and the majority of these animals fail to reach the weight of littermates (20). To determine whether loss of NKCC1 resulted in alterations in the intestinal epithelia, histological analysis was carried out on tissue from all regions of both neonates and adult NKCC1-deficient animals. As can be seen in Fig. 1, D and E, the intestinal epithelium was largely normal. Comparison with wild-type tissue did, however, reveal a slight increase in the frequency of dilated crypts in the NKCC1-deficient tissue.

Jejunum. Ion transport across a midjejunal region was characterized for mice carrying the Slc12a2Delta 1065-1137 and Slc12a2Delta 506-621 mutations. "Basal" refers to the Isc recorded 30 min after mounting. There were no significant differences in either the basal or stimulated Isc between the two Slc12a2 mutations and their respective controls. Therefore, only the jejunal data from the Slc12a2Delta 1065-1137 jejuna (and littermate controls) are shown.

The basal bioelectric properties for the wild-type and -/- jejuna bathed in KBR (as well as 0 HCO3- and 0 Cl-) are shown in Table 1. When bathed in bilateral KBR, the jejuna from the -/- mice exhibited a net basal Isc that did not differ significantly from the control (+/+) jejuna (Fig. 2A; Table 1). The basal Isc (determined at the midpoint of the oscillations) of both the control and the -/- preparations exhibited spontaneous oscillations that have been previously characterized as oscillations in the basal rate of anion secretion mediated by neurotransmitter release from the enteric nervous system (Refs. 7 and 25; Fig. 3, A and C). The magnitude of these oscillations was virtually identical for control and -/- mice (Table 2). The basal Isc of the +/+ jejuna exhibited a small decrease in response to basolateral bumetanide, whereas the basal Isc of the -/- jejuna was unresponsive to bumetanide (Fig. 2B, KBR). The bumetanide-insensitive basal Isc was similar for the two genotypes (Fig. 2B). Bumetanide also significantly reduced the magnitude of the oscillations in the +/+ preparation (see Ref. 7) but had no effect on the oscillations in the -/- preparations (data not shown). Interestingly, the transmembrane conductance in KBR was significantly greater in the -/- jejunum compared with the wild-type preparations (Table 1).

                              
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Table 1.   Jejunal basal bioelectric properties



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Fig. 2.   A: effect of ion-replacement protocol on net basal short-circuit current (Isc) in jejuna. Jejuna were incubated in the indicated buffer for 30 min before the measurements were taken. Open bars, normal tissue; solid bars, NKCC1 -/- tissue. KBR, Krebs-HCO3- Ringer buffer bilateral (n = 14 normal; n = 18 NKCC1 -/-); 0 Cl-, nominally Cl--free bilateral (n = 5 normal; n = 8 NKCC1 -/-). 0 HCO3-, bilateral HCO3--free buffer bilateral (n = 8 normal; n = 9 NKCC1 -/-). 0 Cl-/0 HCO3-, nominally Cl-- and HCO3--free bilateral (n = 7 normal; n = 4 NKCC1 -/-). ** P<= 0.01 vs. respective tissue in KBR. B: effect of bumetanide on basal Isc. Open bars, bumetanide-sensitive component of basal Isc in the +/+ jejuna; hatched bars, bumetanide-insensitive component of basal Isc in the +/+ jejuna; solid bars, bumetanide-insensitive component of basal Isc of the -/- tissue (none of the -/- tissue responded to bumetanide). * P<= 0.05, bumetanide-insensitive Isc vs. basal Isc (bumetanide insensitive) in tissue incubated in KBR for the respective genotype; + P<= 0.05, bumetanide-sensitive Isc significantly different vs. bumetanide-sensitive Isc in KBR and bumetanide-insensitive Isc (+/+) vs. KBR (+/+) jejuna. n = 4 for each group. All data are means ± SE.



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Fig. 3.   A and B: recorder trace of Isc from normal jejunum without and with DIDS application, respectively. C and D: recorder trace of Isc from NKCC1 -/- jejunum without and with DIDS application, respectively. Forsk, 10-5 M forskolin serosal; Bumet, 10-4 M bumetanide serosal; glucose, 10 mM mucosal; DIDS, 10-3 M serosal.


                              
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Table 2.   Magnitude of jejunal Isc oscillations

Most of the preparations in this investigation were studied as full-thickness preparations with the enteric nervous system intact. To determine the role of the enteric nervous system in maintaining the Isc in wild-type and -/- tissue, some preparations were treated basolaterally with TTX. In both the normal and -/- jejuna, TTX caused a significant decrease in the magnitude of the basal Isc but the post-TTX Isc did not differ between the genotypes (Fig. 4A). The post-TTX conductance was again significantly greater in the -/- preparations (data not shown). The influence of bumetanide on the basal Isc was not studied in the TTX-treated preparations.


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Fig. 4.   A: basal and post-TTX Isc in jejunal preparations incubated in KBR or bilateral HCO3--free buffer. * P<= 0.05 vs. basal KBR for respective genotype. ** P<= 0.01 vs. same genotype post-TTX KBR. In KBR-incubated preparations, n = 8 and 9 for +/+ and NKCC1 -/-, respectively; in 0 HCO3--incubated preparations, n = 4 for both genotypes. B: forskolin response of TTX-treated tissue incubated in KBR or bilateral HCO3--free buffer. ** P<= 0.01 vs. +/+ tissue incubated in the same buffer. In KBR-incubated preparations, n = 8 and 9 for +/+ and -/-, respectively; in 0 HCO3--incubated preparations, n = 4 for both genotypes. A and B: open bars, +/+ tissue; solid bars, NKCC1 -/- jejuna. C: effect of bumetanide on the forskolin-stimulated Isc in TTX-treated jejuna. Open bars, bumetanide-sensitive Isc +/+ tissue; hatched bars, bumetanide-insensitive Isc +/+ tissue; solid bars, bumetanide-insensitive Isc -/- tissue. + P<= 0.05, +/+ vs. -/-bumetanide-sensitive Isc in KBR; * P <= 0.05 bumetanide-insensitive Isc -/- vs. +/+ jejuna in KBR; ++ P <= 0.01, bumetanide-sensitive Isc +/+ vs. -/- in 0 HCO3-; ** P <= 0.01 bumetanide-insensitive Isc in 0 HCO3- buffer -/- vs. bumetanide-insensitive Isc -/- in KBR buffer. Sample sizes same as in B. Values are means ± SE.

To identify potential routes for Cl- influx under basal conditions across the basolateral enterocyte membrane, the jejuna of NKCC1 -/- mice were studied using ion-substitution protocols. Bilateral Cl- replacement eliminated the spontaneous oscillations in the basal Isc of both the wild-type and the -/- jejuna (Table 2). In bilateral Cl--free buffer, net basal Isc was significantly and proportionately reduced in both genotypes (Fig. 2A and Table 1), and tissues of neither genotype responded to bumetanide (Fig. 2B, 0 Cl-). As in KBR, the conductance of the -/- preparations was significantly increased compared with the +/+ tissue (Table 1).

In another series of ion-substitution experiments, HCO3- was removed from the buffer bathing both tissue surfaces. HCO3- substitution had no significant effect on the magnitude of the net basal Isc in the control jejuna. However, this maneuver significantly reduced the magnitude of the Isc in the -/- jejuna compared with tissue incubated in KBR (Fig. 2A and Table 1). In HCO3--free buffer, the Isc of the +/+ jejuna had a significantly greater response to bumetanide than did the +/+ preparations in KBR (Fig. 2B). The -/- tissue again was not inhibited by bumetanide in this buffer. Interestingly, bilateral removal of HCO3- significantly reduced the magnitude of the Isc oscillations in both the normal and -/- jejuna (Table 2). The conductance was again significantly greater in the -/- tissue compared with the +/+ preparations in HCO3--free solutions (Table 1).

A number of TTX-treated preparations were studied in bilateral HCO3--free buffer. HCO3--free buffer had no significant effect on the magnitude of the net basal Isc of the normal preparations (post-TTX) compared with normal TTX-treated preparations in KBR (Fig. 4A). However, the post-TTX Isc in the -/- jejuna incubated in HCO3--free buffer was significantly reduced (3-fold) compared with the TTX-treated -/- jejuna incubated in KBR (Fig. 4A).

Removal of both HCO3- and Cl- bilaterally from the solutions bathing the tissues caused a significant reduction in the magnitude of the Isc of the normal tissue (compared with tissue in KBR and HCO3--free KBR but not Cl- free alone) but did not significantly reduce the magnitude of the Isc in the -/- tissue compared with tissues incubated in bilateral HCO3-- or Cl--free medium (Fig. 2A). We did not test the effect of bumetanide on the basal Isc of these tissues. However, because bumetanide had no effect on the basal Isc in the bilateral Cl--free group, this drug would not be expected to have an effect on this group of tissues.

We also used selected drugs to test for other transport paths that could account for basolateral anion influx in wild-type and mutant jejunal epithelia. In the normal jejunum, basolateral DIDS caused a large transient increase in Isc [Delta  = 65.7 ± 2.7 µA/cm2 (n = 7)] (Fig. 3B). After DIDS application, the oscillations in the basal Isc tended to be somewhat smaller than the magnitude of the pre-DIDS oscillations; however, this difference was not significant, and DIDS had no detectable effect on basal Isc [56.6 ± 7.1 vs. 65.5 ± 4.4 µA/cm2 for pre- vs. post-DIDS (5 min); Fig. 3B]. In contrast, in the NKCC1 -/- jejuna, basolateral DIDS caused a small, transient (2-3 min) increase in the Isc (Fig. 3D). Three to five minutes after DIDS application, the oscillations in the basal Isc disappeared (n = 5) (Fig. 3D), and the magnitude of the basal Isc was significantly reduced [74.1 ± 16 vs. 45.3 ± 7.9 µA/cm2 for pre- vs. post-DIDS (n = 5 for both); P<= 0.05].

Mice with mutant NKCC1 exhibited major differences from wild-type mice in response to cAMP-stimulated Cl- secretion. In KBR buffer, the jejuna of the -/- mice responded to forskolin with an increase in Isc that was markedly reduced compared with the forskolin response exhibited by the normal jejuna (Fig. 3, A and C, and Fig. 5A, KBR data). In the +/+ tissue, virtually the entire forskolin response was inhibited by bumetanide, whereas the forskolin response of the -/- jejuna exhibited no significant response to bumetanide (Fig. 3, A and C, and Fig. 5B, KBR data).


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Fig. 5.   A: jejunal Isc response to forskolin in preparations incubated in indicated buffers (see Fig. 1). Open bars, +/+ (normal) tissue; solid bars, NKCC1 -/- jejuna. * P<= 0.05 vs. normal jejuna in KBR; § P<= 0.05 vs. -/-jejuna in KBR. In KBR, n = 10 for both genotypes; in 0 Cl- and 0 Cl-/0 HCO3-, n = 5 and 4 for normal and NKCC1 -/-, respectively; in 0 HCO3-, n = 4 and 5 for normal and NKCC1 -/-, respectively. B: effect of bumetanide on the forskolin-stimulated Isc in jejuna. Open bars, bumetanide-sensitive component of the forskolin-stimulated Isc in +/+ jejuna; hatched bars, bumetanide-insensitive Isc in +/+ preparations; solid bars, bumetanide-insensitive Isc in the -/- jejuna. ** P<= 0.01 bumetanide-sensitive Isc in KBR (+/+) vs. bumetanide-sensitive forskolin response in 0 Cl- buffer (+/+). * P <=  0.05 bumetanide-insensitive response -/- vs. +/+ in KBR. + P<= 0.05 -/-in 0 HCO3-buffer vs. -/-in KBR. In KBR, n= 10 for both genotypes; in 0 Cl-, n = 5 and 4 for normal and NKCC1 -/-, respectively; in 0 HCO3- and 0 Cl-/0 HCO3-, n = 4 for both genotypes.

In TTX-treated preparations bathed in KBR, the magnitude of the forskolin response was also significantly reduced in the -/- jejuna (Fig. 4B). Bumetanide again inhibited the forskolin response in the TTX-treated normal preparations but had no effect on the forskolin response in the TTX-treated -/- jejuna (Fig. 4C). However, in the normal TTX-treated preparations a small bumetanide-insensitive forskolin-stimulated Isc was present (Fig. 4C) that was significantly less than the bumetanide-insensitive response of the -/- tissue, a pattern similar to the forskolin tissues without TTX pretreatment (Fig. 5B).

The response to forskolin was significantly reduced by bilateral Cl- removal in normal tissue, whereas this maneuver had no significant effect on the magnitude of the forskolin response in the -/- jejuna (Fig. 5A). In normal tissue, this was caused by the elimination of the bumetanide-sensitive component of the forskolin response (Fig. 5B).

Removal of HCO3- from the medium had no effect on the magnitude of the forskolin response in the normal jejuna, which was 100% inhibitable by bumetanide (Fig. 5). However, this maneuver significantly reduced the magnitude of the forskolin response (bumetanide insensitive) in the -/-jejuna compared with tissue incubated in KBR (Fig. 5).

TTX-treated preparations incubated in bilateral HCO3--free buffer exhibited the same pattern of bioelectric responses as did non-TTX-treated preparations in this buffer. The magnitude of the forskolin response in HCO3--free buffer in the +/+ preparations was unchanged from that in KBR, whereas the -/- jejuna exhibited a significantly attenuated response (Fig. 4B). The normal preparations (0 HCO3- post-TTX) exhibited a marked bumetanide sensitivity after forskolin (virtually 100% bumetanide sensitive), whereas the -/- preparations did not respond to bumetanide (Fig. 4C).

The forskolin responses of both genotypes were significantly attenuated in Cl--free/HCO3--free buffer compared with the responses exhibited by the tissues in KBR (Fig. 5A). In Cl--free/HCO3--free buffer, the entire forskolin-stimulated Isc was bumetanide insensitive in both groups (Fig. 5B). An additional group of -/- jejunal preparations was studied in basolateral Na+-free buffer and exhibited no significant response to forskolin (Delta Isc = 6 ± 2.7 µA/cm2; n = 4).

Another series of experiments was conducted on jejuna from -/- mice in which only basolateral Cl- or HCO3- was replaced. The data from these experiments did not differ significantly from those obtained from the bilateral solution replacement studies (data not shown).

The magnitude of the forskolin response in DIDS-pretreated tissues (serosal) did not differ significantly from tissue not pretreated with serosal DIDS in either genotype [+/+: Delta Isc with no DIDS treatment 51.7 ± 8.1 µA/cm2 (n = 10) vs. Delta Isc with DIDS pretreatment 50.0 ± 9.7 µA/cm2 (n = 7); -/-: Delta Isc with no DIDS treatment 19.2 ± 3.8 µA/cm2 (n = 10) vs. Delta Isc with DIDS pretreatment 19.8 ± 4.4 µA/cm2 (n = 4)]. When DIDS was applied after forskolin and bumetanide (in KBR) in the normal preparations, the DIDS response did not differ significantly from zero (7.2 ± 4.4 µA/cm2; n = 4). In contrast, DIDS caused a significant inhibition in the stimulated Isc in the -/- preparations (-19.4 ± 4.5 µA/cm2; n = 4). In both control and -/- jejuna, the forskolin increase in Isc was insensitive to serosal DMA (10-5 M) and hydrochlorothiazide (10-4 M) (data not shown; both applied after forskolin).

Cecum. In contrast to the jejuna, the ceca of the NKCC1 -/- mice exhibited basal Isc that were significantly reduced compared with those of ceca from normal mice (Fig. 6 and Table 3). Amiloride (10-4 M, mucosal) had no significant effect on the magnitude of the basal Isc in either the normal or -/- tissue (data not shown). Bumetanide caused a 34 ± 2.9% inhibition of basal Isc in the +/+ ceca, whereas in the -/- ceca, bumetanide had no significant effect on basal Isc (5.6 ± 5.6% increase in basal Isc). The bumetanide-insensitive Isc in the +/+ tissue (55.9 ± 15 µA/cm2, n = 4) was significantly greater than that in the -/- tissue (P < 0.05). Interestingly, unlike the jejuna, in the ceca there was no significant difference in the transepithelial conductance between the two genotypes (Table 3).


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Fig. 6.   Net basal Isc of ceca in the buffers indicated (see Fig. 1 legend). Open bars, normal; solid bars, NKCC1 -/-. ** P<= 0.01 vs. normal in KBR; * P<= 0.05 vs. same genotype in KBR. In KBR, n = 15 and 14 for normal and NKCC1 -/-, respectively; in 0 Cl- and 0 HCO3-, n = 5 and 4 for normal and NKCC1 -/-, respectively; in 0 Cl-/0 HCO3-, n = 4 and 5 for normal and NKCC1 -/-, respectively.


                              
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Table 3.   Cecal basal bioelectric properties

Incubating normal ceca in bilateral Cl--free buffer significantly decreased the basal Isc, whereas this maneuver did not change the basal Isc of the -/- tissue (Fig. 6). The effect of bumetanide on the basal Isc was studied only in preparations incubated in KBR. Incubating normal ceca in bilateral HCO3--free buffer significantly reduced basal Isc compared with KBR (Fig. 6). This maneuver did not significantly reduce the magnitude of Isc in the -/- ceca. Removing both Cl- and HCO3- bilaterally from the medium reduced the magnitude of basal Isc in the normal tissue compared with tissue incubated in KBR (Fig. 6). In this buffer, the basal Isc of the -/- ceca was small (only ~9.4 µA/cm2) and was significantly different from the basal Isc in KBR.

Interestingly, the magnitude of the forskolin response in the -/- ceca did not differ significantly from that of the +/+ ceca (Fig. 7A, KBR data). However, bumetanide inhibited most of the forskolin response in the normal ceca but had no significant effect on the response of ceca from the -/- mice (Fig. 7B, KBR data); thus the bumetanide-insensitive Isc was significantly elevated in the -/- tissue (KBR) compared with normal preparations (Fig. 7B).


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Fig. 7.   A: change in Isc in response to forskolin in ceca incubated in buffers indicated. Open bars, normal; solid bars, NKCC1 -/-. ** P<= 0.01 vs. respective genotype in KBR. In KBR, n = 10 for both genotypes; in 0 Cl- and 0 HCO3-, n = 5 and 4 for normal and NKCC1 -/-, respectively; in 0 Cl-/0 HCO3-, n = 4 for both genotypes. B: effect of bumetanide on the forskolin-stimulated Isc in ceca. Open bars, bumetanide-sensitive component of the forskolin-stimulated Isc in +/+ jejuna; hatched bars, bumetanide-insensitive Isc in +/+ preparations; solid bars, bumetanide-insensitive Isc in the -/- jejuna. **P<= 0.01 bumetanide-sensitive forskolin response in +/+ tissue vs. bumetanide-sensitive forskolin response (= 0) in -/-tissue in same buffer; ++ P <= 0.05 vs. bumetanide-insensitive forskolin response in +/+; § P <= 0.01 vs. bumetanide-insensitive response in KBR for -/-; * P<= 0.05 vs. respective genotype in KBR (bumetanide-insensitive Isc). In KBR, n = 10 for both genotypes; in 0 Cl-, n = 5 and 4 for normal and NKCC1 -/-, respectively; in 0 HCO3-, n = 4 for both genotypes; in 0 Cl-/0 HCO3-, n = 4 for both genotypes.

Ion-substitution studies were again carried out in an attempt to further characterize the response to forskolin in these tissues. Bilateral Cl--free buffer significantly reduced the magnitude of the forskolin response in tissues of both genotypes compared with the respective tissues incubated in KBR (Fig. 7A). Neither tissue responded to bumetanide (postforskolin) in Cl--free buffer (Fig. 7B); therefore, the magnitude of the bumetanide-insensitive forskolin responses was nearly identical in this buffer (Fig. 7B).

The forskolin response was similar to control (KBR) in normal ceca in bilateral HCO3--free buffer, and the ratio of the bumetanide-sensitive vs. bumetanide-insensitive response was similar to that of the +/+ tissues in KBR (Fig. 7). However, in the -/- ceca, the magnitude of the forskolin response was significantly reduced in bilateral HCO3--free buffer compared with KBR (Fig. 7A). The -/- ceca incubated in bilateral HCO3- buffer again did not respond to bumetanide (Fig. 7B). The bumetanide-insensitive Isc was significantly greater in the -/- preparations compared with the bumetanide-insensitive Isc of the normal preparation incubated in the buffer. The effect of bilateral HCO3--free buffer on the magnitude of the forskolin response in TTX-treated ceca was also investigated. The responses of these preparations were similar to those of ceca not pretreated with TTX [forskolin response, HCO3- free, TTX treated +/+Delta Isc, 137.4 ± 17 µA/cm2 (n = 4) vs. -/-, 31.9 ± 2.1 µA/cm2 (n = 4); p<= 0.001; compare with data in Fig. 7A].

When both Cl- and HCO3- were removed bilaterally from the medium, the forskolin response was very small and did not differ between the two genotypes (Fig. 7A). Neither tissue responded to bumetanide when incubated in this buffer (Fig. 7B).

In some preparations (KBR) DIDS was added after forskolin/bumetanide. DIDS caused a significant decrease in the magnitude of the forskolin-stimulated Isc in the -/- ceca only (30.6 ± 7.9 µA/cm2; n = 4; P < 0.05).

Removal of both HCO3- and Na+ from the basolateral side of the -/- ceca (HCO3- was also removed from the mucosal side to ensure that no HCO3- was transported across the tissue) caused no further attenuation in the forskolin response compared with HCO3- removal alone (Fig. 8). The forskolin response of the -/- ceca in this buffer was not sensitive to DIDS (data not shown). However, when Cl- was also removed from the basolateral side of the tissue (in addition to the above ions), the forskolin response was significantly decreased (Fig. 8).


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Fig. 8.   Isc response to forskolin in NKCC1 -/- ceca studied in the buffers indicated. * P<= 0.05 vs. preparations incubated in HCO3-and Na+-free buffer; n = 4 for each group. Buffers are bilateral (m/s) or serosal (s) only.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found that NKCC1 (encoded for by the Slc12a2 gene) mRNA expression was very prominent in the salivary gland and the more distal regions of the intestinal tract (Fig. 1). The Na+-K+-2Cl- cotransporter is located in the basolateral membrane of many secretory epithelial cell types. Although the cotransporter is thought to play a role in basal and stimulated Cl- secretion by transporting Cl- across this barrier (Fig. 9), the basolateral (and apical) membranes of many types of epithelial cells also may contain Cl-/HCO3- exchangers (anion exchanger; AE) that can transport Cl- in exchange for HCO3- (Fig. 9). Na+-HCO3- (NBC)-coupled entry has also been identified on the basolateral membrane of some intestinal epithelia, which can mediate a HCO3- contribution to intestinal anion secretion.


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Fig. 9.   Model of potential basolateral entry paths in an intestinal epithelial cell. As both crypt and villus cells can secrete, this model could represent either cell type. CA, carbonic anhydrase; AE, anion exchanger; NHE, Na+/H+ exchanger; NBC, Na+-HCO3- cotransporter; CFTR, cystic fibrosis transmembrane conductance regulator; DMA, dimethylamiloride.

The NKCC1-deficient mutant mice do not die of intestinal obstruction and rupture either in the early prenatal period or as adult mice (20). They thus differ from CF mice, which have a mutation in the CFTR gene that mediates apical membrane Cl- translocation, and typically exhibit severe intestinal pathophysiology resulting in both neonatal and adult mortality (3, 28). A study was recently published on another NKCC1-deficient mouse model consistent with our finding that there was no increased death rate in neonatal NKCC1 -/- pups (5). However, unlike our study, Flagella et al. (5) reported a relatively high death rate (28%) of the NKCC1 -/- mice in the periweaning period. Although no definite cause of death was given, intestinal bleeding was noted in some pups (5). Whereas CF mice, which are deficient in apical membrane Cl- channel activity, exhibited dilated mucus-filled crypts and intestinal goblet cell hypertrophy and hyperplasia (8), the NKCC1 -/- mice exhibited only very mild intestinal histopathology in the form of scattered dilated crypts that did not appear to contain mucus (Fig. 1, D and E). In CF mice, the intestinal morbidity and high rate of prenatal mortality appear to result from the inability of the intestinal tract to secrete either Cl- or HCO3- in the basal or stimulated state (2, 7, 12, 24). Because our NKCC1 -/- mice exhibit little intestinal histopathology and no obvious morbidity or mortality related to intestinal problems, it seems likely that the intestinal tracts of these animals are capable of sufficient liquid secretion to circumvent the intestinal problems that characterize the CF mice. We therefore characterized the anion transport paths that likely mediate liquid secretion in these animals.

Jejunum. Flux studies as well as ion-replacement studies (7, 25) suggest that a large part of the basal Isc in the normal mouse jejunum reflects Cl- secretion or is Cl- dependent (7, 25). In our study, the basal Isc was insensitive to bumetanide in both the +/+ and -/- jejuna and was of nearly identical magnitude in these genotypes. Blocking neurotransmitter release from the enteric nervous system (TTX-treated tissue) did not appear to alter this relationship. However, Cl- is clearly involved in the maintenance of the basal Isc, because removal of Cl- from the bathing medium significantly reduced the basal Isc similarly in both genotypes.

It is well established (2, 7, 15, 24, 25) that the normal murine intestine is capable of substantial electrogenic HCO3- secretion under both basal and stimulated conditions. Studies have identified an NBC coupled cotransport entry path at the basolateral membrane of enterocytes of several species (Refs. 1, 17; Fig. 9). The NBC has been found to be electrogenic with the usual stoichiometry being 1:2 or 1:3 (23). In addition, the NBCs (there may be a number of isoforms, see Ref. 23 for details) are Na+ dependent, HCO3- dependent, and usually blocked by stilbenes such as DIDS (23). It should be pointed out that some studies have failed to detect this basolateral cotransporter in intestine (10).

HCO3- removal significantly decreased the magnitude of the bumetanide-insensitive Isc in both genotypes. In addition, in the +/+ jejuna, this protocol significantly increased the bumetanide-sensitive component of the basal Isc, leaving the net Isc unchanged. This observation lends support to previous reports that removal of one of the anions (HCO3-) from the buffer increased the secretion of the other (Cl-) (17, 25). In both +/+ and -/- jejuna, one could speculate that in KBR at least part of the basal bumetanide insensitive Isc may be mediated by NBC basolateral entry. In the NKCC1 -/- tissue, the criteria for assigning NBC a role in basal transport appear to be met in that the basal Isc was electrogenic, Na+ dependent (see Table 2), HCO3- dependent, and at least partially sensitive to DIDS. In the -/- jejuna, DIDS completely eliminated the spontaneous oscillations in the basal Isc as well as decreasing the magnitude of the basal Isc. An inhibitory effect of DIDS on the NBC and or AE would be compatible with this observation. If the bumetanide-insensitive Isc in either genotype is due at least in part to HCO3- secretion, then why is the Isc diminished by Cl- removal? An interesting study on isolated colonic crypts reported that basolateral Cl- removal significantly attenuated the magnitude of the stimulated rate of HCO3- secretion in these preparations (6). In pancreatic cells, it has been suggested that Cl- secretion maintains the driving force for basolateral NBC entry by depolarization of the membrane (26). If this mechanism is operating in the jejuna under the conditions of our study (either or both +/+ and -/-), basolateral Cl- entry would have to be via a non-NKCC1 pathway (see Fig. 9). Another possibility is that a parallel operation of a basolateral NBC and AE may been involved. In this scenario, HCO3- taken up via the NBC would be recycled across the basolateral membrane via the AE. Thus net electrogenic Cl- secretion would result. Either or both of these possibilities would explain the need for all three ions (Na+, Cl-, and HCO3-; Table 2).

Interestingly, the transepithelial conductance of the NKCC1 -/- jejuna was significantly greater than that of the normal preparations in every buffer studied. The reason for this result is not known. As >80% of the transepithelial conductance of intestinal epithelia has been attributed to the paracellular pathway, the increased conductance most likely reflects a change in the conductance of this pathway (see Ref. 21 for discussion). It has been shown that when intestinal cells are stimulated to secrete, there is up to a 50% decrease in conductance (21). It has been suggested that this change reflects cell swelling, which in turn reduces the width of the intracellular space, thus decreasing conductance (21). If NKCC1 played a role in cell volume and the volume of the NKCC1 -/- cells was decreased compared with normal (unstimulated) cells, this difference theoretically might increase the transepithelial conductance. In our normal intestine, bumetanide (before forskolin) had no significant effect on the transepithelial conductance (data not shown). Therefore, at least on an acute basis, blocking the NKCC1 pathway in normal jejuna does not increase transepithelial conductance. Further study will thus be necessary to explain the difference in the transepithelial conductance across the jejuna of the two genotypes.

Removing both Cl- and HCO3- from the medium did not further diminish the magnitude of the basal Isc in -/- jejuna compared with the Isc when either anion was present. The origin of the Isc in bilateral Cl-/HCO3--free medium in the +/+ and -/- jejuna is not known. It would not be expected to be amiloride-sensitive Na+ absorption because this transporter is not present in jejunal tissue. Also, because there was no apical glucose present, electrogenic Na+-glucose cotransport could not explain the basal Isc.

In the jejunum of the normal mouse, forskolin induced an increase in Isc that was Cl- dependent. The inhibition of the forskolin response by bumetanide, the marked decrease in the magnitude of this response when tissues were incubated in bilateral Cl--free buffer, and the failure of bilateral HCO3--free buffer to change the magnitude of this response support the speculation that the forskolin response reflects Cl- secretion. Therefore, NKCC1 appears to play a major role in the forskolin-stimulated response in this tissue.

It is interesting to note that there is a significant bumetanide-insensitive response to forskolin in the normal preparations in 0 Cl- buffer (12.4 ± 4.2 µA/cm2) and that this response did not differ in magnitude from the -/- jejuna in the same buffer. It is likely that the same basolateral entry mechanism supports the bumetanide-insensitive forskolin response in 0 Cl- buffer in normal and -/- jejuna.

The forskolin response was significantly reduced in the jejuna of the NKCC1 -/- mutant mice and was bumetanide insensitive. The -/- jejunal forskolin response in TTX-treated tissues was also significantly reduced compared with normal TTX-treated tissue (KBR). In a recent study on another NKCC1-deficient mouse model, some intestinal ion-transport data were reported (5). Flagella et al. (5) also noted a reduced response to forskolin (bumetanide insensitive) in the NKCC1 -/- jejunum. In our investigation, removal of Cl- from the bathing solution did not alter the magnitude of the response. Therefore, basolateral Cl- entry via a Cl-/HCO3- exchanger does not appear to play a role in the forskolin response. Removal of HCO3- from the buffer of the NKCC1 -/- jejunum, however, did significantly decrease the magnitude of this response to forskolin. In addition, removal of basolateral (but not apical) Na+ completely eliminated the forskolin response in the -/- tissue. These data suggest that the forskolin response by the NKCC1 -/- jejuna is dependent on basolateral Na+/HCO3- entry. If at least a portion of the basal Isc and the forskolin response are due to HCO3- secretion, it is not readily apparent why the basal Isc is Cl- dependent whereas the forskolin response is not. Interestingly, in the rabbit mandibular gland, when Cl- secretion was blocked, HCO3- was able to support salivary secretion, but at a reduced rate (19). Novak and Young (19) suggest that the lower rate of HCO3- secretion was due only to the anion being at a lower concentration than Cl-.

There is much variability in the literature regarding the ability of DIDS to block Cl-/HCO3- (14, 29) and Na+-HCO3- (4, 22, 27) entry pathways in the intestine. Interestingly, one study found that DIDS blocked the vasoactive intestinal polypeptide- or PGE2-stimulated HCO3- secretion in the rabbit duodenum, but when HCO3- secretion was induced with dibutyryl-cAMP the HCO3- secretory response was no longer sensitive to DIDS (31). In our jejunal preparations incubated in KBR, basolateral DIDS applied after bumetanide and forskolin was without effect in the +/+ tissue but significantly inhibited the forskolin response in the -/- jejuna. Interestingly, DIDS applied before forskolin was without effect on the magnitude of the forskolin response in either genotype. The reason for these differences is unknown. However, the data obtained when DIDS was applied after forskolin support the ion-substitution studies that suggest that the basolateral NBC may play a role in the stimulated secretory response in the -/- jejuna.

Cecum. The normal ceca exhibited a significantly greater net basal Isc than did the -/- tissue. This difference was primarily due to the significant bumetanide-sensitive component of the basal Isc in the normal cecum, which was absent in the normal jejunum. In addition, because removal of either Cl- or HCO3- significantly reduced the net Isc in the normal ceca, it is likely that the basal Isc in this region may reflect a combination of HCO3- basolateral entry (probably by NBC) and Cl- secretion, basolateral entry via NKCC1.

In the ceca of the -/- mice, the basal Isc was significantly reduced compared with the +/+ tissue in KBR and was unresponsive to bumetanide. The low basal Isc (~30 µA/cm2) in the -/- ceca was not significantly altered by removal of either Cl- or HCO3- but was significantly decreased when both anions were removed from the bathing medium. Although the ion-transport path(s) responsible for this small basal Isc in these preparations is unknown, the absence of a response to apical amiloride (in +/+ or -/- preparations) suggests that the Isc is not a result of Na+ absorption. As 1 mM acetazolamide was included in the HCO3--free/CO2-free medium to prevent endogenous HCO3- production, it is unlikely that the tissue generates sufficient HCO3- endogenously to supply HCO3- for secretion when either (or both) Cl- or HCO3- is removed from the medium.

The forskolin response in the -/- ceca, unlike the -/- jejuna, was not significantly reduced compared with that of the normal ceca. The large forskolin response in the -/- ceca was completely insensitive to bumetanide. Thus it appears that in the -/- ceca this pathway is upregulated (~100 µA/cm2) compared with the small bumetanide-insensitive component (~30 ± 12.3 µA/cm2) of the forskolin response in the normal ceca. As in the normal ceca, the response was significantly inhibited by bilateral Cl- substitution, suggesting that Cl- plays a role in the secretory response by a NKCC1 cotransporter-independent pathway. In contrast, in the normal ceca, the majority of the forskolin response appears to be Cl- secretion mediated by basolateral NKCC1 entry. In the NKCC1 -/- ceca, bilateral HCO3--free buffer also significantly reduced the forskolin response to a level not significantly different from that of the preparations incubated in Cl--free buffer. These data suggest that, unlike the normal ceca, the forskolin response is dependent on both Cl- and HCO3- in the NKCC1 -/- ceca. One could postulate that, in the NKCC1 -/- ceca, both an Na+-HCO3--coupled entry path and a Cl-/HCO3- exchanger are expressed at the basolateral membrane and play a role in the forskolin-stimulated secretory response (as was suggested for the jejuna). DIDS significantly inhibited the forskolin response in the -/- ceca (in KBR), consistent with this notion.

In the absence of both HCO3- and basolateral Na+, a substantial (~50 µA/cm2) forskolin response (~50% of the forskolin response in KBR) persisted in the -/- ceca. The forskolin response in these tissues was sustained (for >20 min) and was not sensitive to DIDS (0 HCO3-, 0 Na+). The forskolin response likely reflected Cl- secretion because removal of basolateral Cl- from the buffer (also bilateral HCO3- free and basolateral Na+ free) reduced the forskolin response to nearly zero (see Fig. 8). Because no HCO3- (or CO2) was present (and 1 mM acetazolamide was included in the buffer), it seems unlikely that sufficient endogenous HCO3- would be generated to activate a basolateral Cl-/HCO3- (DIDS insensitive) exchanger or that sufficient endogenous HCO3- would be produced for apical secretion. Thus we are left with no ready explanation for the DIDS-insensitive, Cl--dependent forskolin response in the NKCC1 -/- ceca. A novel basolateral (Cl-) entry path cannot be ruled out.

In conclusion, the absence of the basolateral NKCC1 transport protein responsible for basolateral cellular Cl- entry appears to cause no gut-related morbidity or mortality in NKCC1-deficient mice. This result contrasts with data from CF mice, which demonstrated that the absence of an apical anion conductance leads to profound gut pathology and death (7). Interestingly, Trout et al. (30) have shown that in porcine bronchial submucosal glands a significant fraction of the liquid secreted by the glands in response to ACh was inhibited by bumetanide. Inhibiting Cl- secretion alone did not cause mucus accumulation in the glands (13). However, when both Cl- and HCO3- secretion were blocked, the glands became plugged with mucus. Thus, as long as either HCO3- or Cl- secretion was intact, no apparent submucosal gland pathology resulted. Comparison of the phenotypes of the CF and NKCC1 -/- mice suggests that a similar conclusion pertains to the gut. In the CF mouse, the absence of CFTR results in a failure to secrete Cl- or HCO3- (basal or stimulated), and severe gut pathology results. In the NKCC1 -/- intestine, the presence of basal and stimulated HCO3- secretion as well as a component of Cl- secretion reflecting non-NKCC1 basolateral entry likely protects the gut from obstruction/injury. Further study, e.g., radioisotope flux and pH-stat measurements, will be necessary to provide direct evidence that HCO3- secretion, or another non-NKCC1 basolateral entry pathway, is relatively upregulated in the NKCC1 -/- intestine.


    ACKNOWLEDGEMENTS

This study was supported by National Intitutes of Health Grants SCOR 1-P50 HL-60280-01 and PPG 5-P01-HL-34322, and Cystic Fibrosis Foundation RDP R026 (Project 14).


    FOOTNOTES

Address for reprint requests and other correspondence: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7011 Thurston-Bowles Bldg., CB# 7248, The Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: bgrubb{at}med.unc.edu).

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.

Received 3 December 1999; accepted in final form 20 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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