Ion transport across the normal and CF neonatal murine intestine

B. R. Grubb

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neonatal mice with cystic fibrosis (CF) exhibit a very high mortality due to intestinal obstruction localized primarily to the ileum and colon. It has been hypothesized that lack of Cl- secretion and possibly elevated Na+ absorption contribute to the gut problems in CF neonates. Therefore, intestines (ileum, proximal colon, and distal colon) from normal and CF day-old mouse pups were studied on ultra-small-aperture (0.0135 cm2) Ussing chambers. All three regions of the normal neonatal intestine responded to forskolin with an increase in short-circuit current, which was completely absent in the CF intestine. The neonatal distal colon exhibited a high rate of amiloride-sensitive electrogenic Na+ absorption, which did not differ between the normal and CF preparations. The ileum and proximal colon of both genotypes exhibited a small but significant electrogenic Na+ absorption. The neonatal proximal colon and ileum also exhibited electrogenic Na+-glucose cotransport, which was significantly greater in the normal compared with the CF ileum. In addition, all three intestinal regions exhibited electrogenic Na+-alanine cotransport, which was significantly reduced in two of the regions of the CF neonatal intestine. It is speculated that: 1) the reduced rate of Na+-nutrient cotransport in the CF intestine contributes to the lower rate of growth in CF pups, whereas 2) the elevated electrogenic Na+ absorption in the neonatal intestine, coupled with an inability to secrete Cl-, contributes to the intestinal obstruction in the CF pups.

sodium absorption; calcium secretion; colon; ileum; mice; nutrient uptake; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NEONATAL MAMMAL FACES special challenges with respect to Na+ homeostasis, as large quantities of sodium are needed to accommodate the rapidly expanding extracellular volume. Because the neonate consumes milk relatively low in Na+ and experiences a high renal loss of Na+, the intestines may play a relatively more important role in neonatal Na+ homeostasis than in the adult, with a more efficient kidney (17). Although intestinal absorption is vitally important for growth and volume expansion in the neonate, the importance of intestinal Cl- secretion in the neonate has recently been shown in the various mouse models lacking the cAMP-activated Cl- channel [cystic fibrosis transmembrane conductance regulator (CFTR)]. In most of these CF mouse models, the intestinal tract cannot secrete Cl-, which is associated with the observation that a large number of CF pups die as neonates from intestinal obstruction and rupture (see Ref. 14 for a review). Therefore, studies of both Na+ and Cl- transport across the neonatal intestine may give insight into how normal neonates balance Na+ absorption and Cl- secretion and give clues as to why neonatal CF mice experience such a high rate of intestinal obstruction.

There are few studies on ion transport across the neonatal intestine. In the rat, due to the inability to study guts of smaller neonates, most studies have focused on pups that were greater than 8 days old. With the exception of the limited data that have recently been published on the colon of the Na+ channel (alpha ) knockout neonatal mouse (15), there are no published data on ion transport across the neonatal murine intestine. With the emergence of transgenic and knockout mouse models, many of which exhibit a neonatal lethal phenotype, study of the neonatal (and prenatal) tissue may offer the only opportunity to test for alterations in ion transport rates. Therefore, it is important to have techniques to study ion transport across neonatal murine tissue.

In the present investigation, freshly excised tissue was mounted in miniaturized Ussing chambers to measure the magnitude of electrogenic Na+ absorption and Cl- secretion in the normal neonatal (~24-h-old) murine colon (proximal and distal) and ileum. For comparison, ion transport across the same regions of the neonatal murine CF intestine was investigated to provide clues as to why these regions exhibit such a high incidence of impaction and rupture in the neonatal CF mouse.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice and mounting techniques. The mice used in this study were from a mixed background: BAL/C, DBA/2, C57BL/6, and 129/SVEV. Neonatal mouse pups remained with the mother until the time of the study (12-48 h, averaging 24 h postbirth), and they were then killed by an overdose of CO2. A small piece of tail was clipped, and the genotype of each pup was determined at a later time by PCR analysis of tail DNA (27); thus the study was done blinded. Pups were either heterozygous for CFTR (referred to as normal) or homozygous CFTR(-/-) (cftrtm1unc; referred to as CF). The mean body mass of the normal pups was 1.65 ± 0.7 g (n = 18), which did not differ significantly from the CF pups (1.54 ± 0.1 g; n = 8).

The abdomen was opened with fine scissors, and the designated region of the gut was excised. The intestine was carefully placed on top of a piece of tissue paper (prewetted with buffer, see Solutions and drugs), which covered a small Parafilm O ring seated over the aperture of the Ussing chamber (aperture opening 0.0135 cm2). Then, with the aid of a dissecting scope, the small piece of intestine (2-4 mm long) was split longitudinally using a pair of fine scissors, and any intestinal contents were removed with forceps. The split piece of intestine (apical side up) was positioned over the chamber aperture by moving the piece of tissue paper on which it resided over the chamber aperture, which was illuminated by a light from below. Another Parafilm O ring was placed on the apical surface of the intestine, and the two chamber halves were fitted together. The intestine of the neonatal mouse (until about day 5) is very delicate. However, with care, virtually 100% of the preparations were mounted successfully. After each hemichamber was filled with buffer, the chambers were interfaced to bath circulators and an electrometer, and the tissue was allowed to equilibrate for 30 min, after which basal bioelectric data were obtained. Details of the Ussing chamber techniques are as previously described for the intestine of the adult mouse (11). Unless indicated otherwise, all drugs were added sequentially in the following order: amiloride, carbachol, forskolin, alanine, glucose.

Solutions and drugs. The tissues were bathed bilaterally (10 ml/side) in Krebs bicarbonate buffer having the following composition (in mM): 140 Na+, 120 Cl-, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO2-4, 0.4 H2PO-4, and 25 HCO-3. Glucose (5 mM) was added to the basolateral side of all tissues as substrate, and 5 mM mannitol was added to the apical side to maintain osmolarity. When glucose (or alanine) was added apically, an equal quantity of mannitol was added basolaterally. In some experiments, tissues were bathed bilaterally with nominally Cl--free bicarbonate buffer (referred to as 0 Cl- buffer in text) in which 115 mM sodium gluconate replaced the NaCl, MgSO4 (1.2 mM) replaced MgCl2, and CaCl2 (1.2 mM) was replaced by calcium gluconate (6 mM calcium gluconate was added to overcome the Ca2+ chelating effects of gluconate). Bumetanide (10-4 M) and carbachol (10-4 M) were added to the basolateral side of the tissue, forskolin was added bilaterally (10-5 M), and all other drugs were added apically. All drugs and chemicals were obtained from Sigma (St. Louis, MO).

Statistics. All data are expressed as means ± SE. A Student's t-test was used to compare two groups.


    RESULTS
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Basal bioelectrics. Despite the small exposed surface area of the neonatal intestinal preparations, measurable and stable bioelectric parameters were obtained. Typical records of the short-circuit current (Isc) for a normal and CF distal colon are shown in Fig. 1. The basal bioelectric data obtained from the three intestinal regions studied for normal and CF preparations are shown in Table 1. For the distal and proximal colonic preparations, both the Isc and potential difference (PD) were significantly lower in the CF preparations. The tissue resistance (Rt) was significantly lower in the CF proximal colon and ileum compared with that of the respective tissues of the normal neonates.


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Fig. 1.   Recorder traces of normal (A) and cystic fibrosis (CF; B) neonatal distal colons. Where indicated, amiloride (Amil) was added (10-4 M) apically, forskolin (Forsk; 10-5 M) bilaterally. Bumetanide (Bumet) was also added (10-4 M, serosally) to normal preparations. Isc, short-circuit current.


                              
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Table 1.   Basal bioelectric measurement of neonatal intestine

Forskolin response. Forskolin elevates intracellular cAMP levels in intestinal cells and in normal mouse intestine induces Cl- secretion through the CFTR Cl- channel (2, 13). Bilateral forskolin addition (10-5 M) induced a significant increase in Isc in all three intestinal regions from the normal neonates (Figs. 1A and 2). In the normal neonatal intestine, the forskolin response is primarily a Cl- secretory response. [In bilateral Cl--free buffer, the magnitude of the response (distal colon) is reduced to 6.5 ± 1.2 µA · cm-2 (n = 7, compare with Fig. 2), and in bilateral Cl--free, HCO-3-free buffer, the forskolin response was found to be 0 ± 0, n = 4; see Ref. 11 for details of buffer composition.] In the CF neonatal intestine, forskolin was completely without effect in all three intestinal regions studied (Figs. 1B and 2).


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Fig. 2.   Change in Isc in response to 10-5 M forskolin (bilateral). Open bars are normal tissue, and solid bars are CF tissue; n = 8, 9, and 11 for normal distal colon, proximal colon, and ileum, respectively; n = 5, 4, and 5 for CF distal colon, proximal colon, and ileum, respectively. ** P <=  0.01, normal vs. CF for each respective region. In all cases, tissues were pretreated with amiloride (10-4 M, apically) before forskolin was added.

Carbachol response. In all three intestinal regions of the normal neonate, the Ca2+-mobilizing agent carbachol (10-4 M basolateral) induced a significant change in Isc that resulted in a large positive Isc (Fig. 3, A and C). In the normal neonatal intestine bathed with Krebs buffer, bumetanide induced a decrease in the carbachol-stimulated Isc, consistent with a Cl- secretory response (Fig. 3, A and D). To test further whether the change in Isc reflected in part a Cl- secretory response, the normal neonatal distal colon was bathed in Cl--free buffer. Under these conditions, carbachol failed to induce an increase in Isc (Fig. 4).


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Fig. 3.   Recorder trace of Isc from normal distal colon (A) and CF distal colon (B), showing tissue response to serosal carbachol (Carb; 10-4 M) addition. C: change in Isc in response to carbachol (10-4 M serosally). * P < 0.01, normal vs. CF tissue. D: bumetanide response of normal and CF distal colons. Distal colons were first treated with amiloride (10-4 M, apically), then carbachol (10-4 M, serosally), and finally bumetanide (10-4 M, serosally). Open bars represent normal tissue, and solid bars represent CF tissue. In all cases, amiloride (10-4 M, apically) was added before carbachol. Sample sizes are as in Fig. 2 except in D, where n = 4 for normal tissue and n = 3 for CF.



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Fig. 4.   Change in Isc in response to carbachol in distal colon. Tissues were incubated for 1 h in Cl--free buffer. Then they were treated with amiloride (10-4 M, apically) followed by carbachol (10-4 M, serosally); n = 7 for normal tissue, and n = 6 for CF tissue. * P <=  0.05, normal vs. CF tissue.

As expected, there was no significant response to carbachol in the CF ileum and proximal colon (Fig. 3C). However, surprisingly, the neonatal CF distal colon responded to carbachol with a significant change (P <=  0.01) in Isc that did not differ in magnitude from normal mice (Fig. 3, B and C). The carbachol-induced change (which was in the positive direction) was characterized by a return of the Isc (postamiloride) toward zero (Fig. 3B). Although this change in Isc in the CF distal colon could reflect secretion of an anion (Cl- or HCO-3), this did not seem likely, as the CF tissue expresses no CFTR to secrete Cl- or HCO-3, nor have alternative Ca2+-mediated Cl- channels been convincingly demonstrated in CF mouse intestine (see DISCUSSION).

The data suggest that the CF neonatal distal colons are secreting K+ (as evidenced by the large residual Isc of reversed polarity postamiloride). In the CF distal colon, this K+ secretion may be inhibited by carbachol, thus bringing the Isc back close to zero (see DISCUSSION). To identify the origin of the carbachol-stimulated Isc in the CF distal colons, neonatal distal colons from CF mice were studied in bilateral Cl--free buffer. The increase in Isc in response to carbachol persisted in CF distal colons in the Cl--free buffer (Fig. 4), suggesting no contribution of Cl- to this response. Also, adding bumetanide after carbachol in the CF distal colon (added to three preparations) caused a positive change in the Isc, which tended to bring the Isc back closer to zero (Fig. 3, B and D). It should be noted that the magnitude of the carbachol response in the CF distal colon is reduced in bilateral Cl--free buffer (Fig. 4) compared with that in Krebs buffer (Fig. 3C). It may be that the Na+-K+-2Cl- cotransporter plays a role in basolateral K+ entry for the K+ secretory response (suggested by the bumetanide data). In the absence of Cl-, basolateral K+ entry through this cotransporter would be eliminated, and thus K+ secretion may be diminished. As more neonatal CF pups become available, K+ channel blockers could be used to characterize the carbachol response further.

Amiloride response. Amiloride was used to estimate the fraction of the basal Isc that reflects electrogenic Na+ absorption. The distal colonic epithelia of both genotypes exhibited a large response to amiloride (Fig. 1). In the distal colon of both genotypes, the entire basal Isc was inhibited by amiloride, and the polarity of the basal Isc was reversed with drug treatment (Figs. 1 and 5A). The magnitude of the amiloride-sensitive Isc did not differ between the two genotypes (Fig. 5A). Although the residual (postamiloride) Isc exhibited by the CF distal colons was about threefold greater (more negative) than that of the normal distal colons, this difference did not reach statistical significance (P = 0.09). The proximal colons from both groups of mice exhibited small but significant responses to amiloride, which were similar for the two groups (Fig. 5B). Likewise, the ileal tissue exhibited a small response to amiloride that did not differ between the CF and normal tissue (Fig. 5B). (Note scale difference compared with Fig. 5A.)


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Fig. 5.   A: amiloride-sensitive Isc (portion of Isc inhibited by amiloride) for normal (open bars) and CF (solid bars) neonatal distal colons. Residual Isc is portion of Isc remaining after amiloride administration. For both genotypes, Isc reversed in polarity following amiloride administration; n = 11 normal, n = 5 CF preparations. B: amiloride-sensitive Isc in proximal colon and ileum of normal (open bars) and CF (solid bars) preparations; n = 9 and 11 for normal proximal colon and ileum, respectively, and n = 4 for CF proximal colon and ileum.

Electrogenic Na+-glucose cotransport. In some of the regions of the mammalian gut (primarily the jejunum), apical addition of glucose elicits a significant increase in Isc due to electrogenic Na+-glucose-coupled absorption (37). In the neonatal proximal colon and ileum of both genotypes, addition of 10 mM apical glucose elicited a significant increase in Isc (Fig. 6). It appears that in the neonatal gut the magnitude of this electrogenic response decreases from proximal to distal regions of the intestine. In the neonatal CF ileum, the response was significantly reduced compared with that in the normal ileum (Fig. 6). In all tissues, the glucose-stimulated Isc was inhibited with phloridzin (10-4 M apical, data not shown).


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Fig. 6.   Change in Isc in response to 10 mM apical glucose addition. Tissues were first pretreated with amiloride. Open bars represent normal tissue, and solid bars represent CF tissue; n = 11, 9, and 11 for normal distal colon, proximal colon, and ileum, respectively; n = 4 for CF distal colon and ileum, and n = 3 for CF proximal colon. * P <=  0.05, normal vs. CF ileum.

Electrogenic Na+-alanine cotransport. Intestinal tissues also possess a number of different transport systems for amino acids, which reflect differences in the physicochemical properties of amino acids (10). Several of the transport systems couple apical amino acid entry to Na+ entry, inducing an electrogenic response. The entry of the neutral amino acid alanine uses such an entry system, with apical addition of alanine inducing an increase in Isc. All intestinal regions studied exhibited a significant increase in Isc in response to 10 mM apical alanine addition (Fig. 7). Again, the intestines exhibited regional differences, with the proximal regions exhibiting the larger response. In all CF tissues, the magnitude of response was approximately one-half to one-third that seen in normal tissue (Fig. 7).


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Fig. 7.   Change in Isc in response to apical alanine addition. Tissues were first pretreated with amiloride (10-4 M, apically), then 10 mM alanine was added apically. Open bars represent normal tissue, and solid bars represent CF tissue; n = 9 for normal distal and proximal colon, n = 10 for normal ileum, and n = 3 for each of the CF tissues. * P <=  0.05, normal vs. CF for respective tissue types.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The capacity for intestinal Cl- secretion appears to be present at birth. Robust Cl- secretory responses have been reported in normal neonatal rat intestine (21). In the normal neonatal murine intestine, Cl- (and possibly HCO-3 to a much lesser extent) secretory responses to forskolin were detected in all three regions of intestine studied (Fig. 2). Interestingly, it has been reported that CFTR mRNA was not detected by Northern blot in the small intestine of the 8-day-old mouse pup (8). The jejuna of the normal newborn mouse does respond to forskolin with a secretory response (unpublished observation). However, the forskolin response in the normal neonatal mouse intestine may be reduced compared with the adult (12). As expected, and as previously reported for all intestinal regions of the adult CF mouse (14), the neonatal CF intestine was completely unresponsive when stimulated by an agent that elevates cAMP (forskolin) and induces Cl- secretion via CFTR (Fig. 2).

Much of the published data suggest that intestinal epithelia possess only one apical Cl- channel, CFTR (1, 3, 19). However, this is still controversial and some groups suggest that intestinal epithelia may possess a separate apical Ca2+-mediated Cl- secretory pathway (26, 35). In intestinal tissue with functional CFTR, agents that increase intracellular Ca2+ can induce Cl- secretory responses. It is thought that the mechanism is an agonist-induced increase in intracellular Ca2+, which activates a basolateral K+ conductance, hyperpolarizes the cell membrane, and increases the driving force for Cl- secretion via CFTR (1, 3). Thus, in the absence of CFTR in intestinal epithelia, Ca2+-mobilizing agents would not be expected to induce Cl- secretion. In all regions of the normal neonatal mouse intestine studied (Fig. 3), carbachol, a Ca2+-mobilizing agent, induced Cl- secretion (29, 33). It has been previously reported that, in the normal adult mouse distal colon and jejunum, carbachol and bethanachol also induce a Cl- secretory response (5, 12).

The neonatal CF ileum and proximal colon were, as expected, completely unresponsive to carbachol (Fig. 3C). However, in the distal colon of the neonatal CF mouse carbachol induced an increase in Isc that did not differ significantly in magnitude from that of the normal neonatal distal colon (Fig. 3, B and C). It is speculated that this response in CF distal colon is most likely due to a carbachol-induced decrease in basal K+ secretion. This inhibitory response could be mediated by the same intracellular signaling mechanism by which carbachol induces Cl- secretion (29), i.e., the Ca2+-induced increase in basolateral K+ conductance hyperpolarizes the apical membrane, thereby reducing the driving force for K+ secretion.

For this hypothesis to be tenable, the distal colon must exhibit basal K+ secretion. The aldosterone levels in neonatal mice have been reported to be very high (6), and the distal colon of the adult aldosterone-stimulated mouse exhibits electrogenic K+ secretion (13). Others have also reported electrogenic K+ secretion by the distal colon of aldosterone-stimulated rats (32). Furthermore, the neonatal rat distal colon, subjected to high aldosterone levels, exhibits a rate of electrogenic K+ secretion that is significantly greater than that seen in the adult distal colon (22). In the aldosterone-stimulated CF adult murine colon, the rate of electrogenic K+ secretion exceeds that of normal mice and the reversal of the polarity of Isc after amiloride treatment reflects the component of electrogenic K+ secretion (13). In the CF neonates, the postamiloride residual Isc was ~70% greater (more negative) than that exhibited by the normal distal colons. However, this difference was not significant. K+ secretion likely accounts for the reversal of Isc polarity in the amiloride-treated normal and CF neonatal distal colons (Fig. 5A). Further evidence that the CF neonatal distal colon secretes K+ comes from the response to bumetanide. Bumetanide increased the magnitude of Isc (postcarbachol) in the CF distal colons (Fig. 3, B and D), reflecting the drug-induced inhibition of electrogenic K+ secretion (5, 22). The Cl--dependent response in the normal neonatal distal colon suggests that Cl- secretion masks this component of the carbachol response in these tissues. The failure of the proximal colon and ileum of the CF neonate to exhibit the same carbachol response as seen in the distal colon probably is due to the low or absent spontaneous K+ secretion in these intestinal regions, even when aldosterone stimulated (13).

The distal colon of the newborn mouse (24 h old) exhibited a substantial amiloride-sensitive Na+ absorption (Figs. 1 and 5). We have previously reported that the preterm (fetal) murine distal colon (~24 h before birth) also exhibited a significant amiloride-sensitive Isc (15). The magnitude of the fetal amiloride response was less than that exhibited by the newborn distal colon, which likely correlates with the higher aldosterone level in the neonatal pups (6) In the neonatal mouse, plasma aldosterone levels begin to rise significantly 2 days before birth and peak at birth, remaining well above the adult level for up to 2 wk after birth (6). In contrast to the neonatal distal colon, the distal colon of the adult mouse on a normal diet exhibits no or very little electrogenic amiloride-sensitive Na+ absorption (5, 13), likely reflecting the low levels of aldosterone in adult mice on a normal diet (13). In all species examined, including the human, electrogenic colonic Na+ absorption appears to correlate with a dramatic increase in serum aldosterone in the perinatal period (9, 17, 20, 23). In contrast to the distal colon, it has been reported that the neonatal human kidney is insensitive to amiloride (17, 25). Similar findings have been reported for the neonatal rat kidney (28), adding support to the hypothesis that, in the neonate, Na+ absorption by the colonic epithelia may play a relatively more important role in Na+ homeostasis than in the adult.

In the present study, there was no significant difference in the magnitude of the amiloride-sensitive Isc in the distal colon of normal and CF pups. These findings contrast with those from the aldosterone-stimulated adult mouse, in which we found that the CF distal colonic epithelia exhibited a significantly greater rate of electrogenic Na+ absorption than did normal colonic epithelia (13).

The proximal colon of neonatal mice exhibited a small but detectable amiloride-sensitive Isc that did not differ between the genotypes (Fig. 5). This finding contrasts with data from adult mice on a normal diet in which neither normal nor CF proximal colons exhibited an amiloride-sensitive Isc (13). In normal adult mice on a low-Na+ diet (high aldosterone), the proximal colon still exhibited no amiloride-sensitive Na+ transport (13), a finding similar to that reported for the adult rat (36). In contrast, the proximal colon of adult CF mice on a low-Na+ diet exhibited amiloride-sensitive electrogenic Na+ absorption (13).

The neonatal murine ileum from both CF and normal mice exhibited a small but measurable amiloride-sensitive Isc that did not differ between the two genotypes. Neither normal nor CF adult murine ileum (normal diet) exhibits a detectable amiloride-sensitive Isc (unpublished observations). No data are available on the amiloride response of the adult CF or normal mouse ileum stimulated by high levels of aldosterone.

In summary, in the neonatal mouse, it appears that amiloride-sensitive Na+ absorption is upregulated in the intestine in both the normal and CF pups compared with adult mice on a normal diet. This undoubtedly is primarily due to the high aldosterone levels measured in neonatal mice.

Electrogenic Na+-coupled glucose absorption is an important means of intestinal uptake of both Na+ and glucose. Although this cotransporter is thought to be principally expressed in the small intestine in adult mammals, the neonatal intestine appears to have a more widespread expression of functional cotransporter activity. Both the proximal colon and ileum of the newborn mouse exhibited electrogenic Na+-glucose cotransport (Fig. 6). In preterm and 2-day-old rat distal colon, glucose increased rectal PD, suggesting the presence of a functional Na+-glucose cotransporter at this site (24). However, after day 2, the response was lost (24). Similar findings were reported for the proximal colon of the newborn pig (16). The importance of the brush-border Na+-glucose cotransporter in the human neonate is evidenced by the profuse, fatal diarrhea that results from a mutation in the gene coding the cotransporter proteins (SGLT1) (37).

In the ileum of the neonatal CF mouse, Na+-glucose transport was found to be significantly less than in the normal neonatal ileum (Fig. 6). The data for adult CF mouse jejunal Na+-glucose transport relative to normal are conflicting. It has been reported that there was no significant difference in Na+-glucose cotransport between normal and CF adult intestine (11, 34). De Jonge et al. (7), however, have reported that in the adult CF murine jejunum Na+-glucose cotransport is decreased.

Electrogenic Na+-amino acid cotransport is primarily expressed in the small intestine (16). Interestingly, the absorptive capacity for amino acid has been found to be greater in the distal part of the small intestine (ileum) than in the more proximal region (10). Neonatal rabbit ileum (4) and porcine colon (16) have been reported to exhibit Na+-amino acid cotransport. Our data also indicate that the ileum of the normal neonatal mouse exhibits electrogenic Na+-alanine cotransport (Fig. 7). We also found that the neonatal CF intestine exhibited a significantly lower rate of Na+-alanine cotransport in the proximal and distal colon compared with normal intestine (Fig. 7).

In the rodent much of the Na+ that is absorbed by the intestine is coupled with either glucose or amino acids (21). The significantly decreased rate of Na+-glucose and -alanine cotransport in the CF intestine (especially if a similar decrease is reflected in the neonatal CF jejunum) may be one explanation as to the significantly slower weight gain in the CF pups compared with their normal counterparts (27). The mechanism by which Na+-nutrient cotransport in the neonatal intestine is diminished is not known. It has been reported that inflammation diminishes Na+-nutrient cotransport in the rabbit ileum (30, 31). Histological examination of neonatal CF intestine revealed no inflammation. However, newborn CF mouse pups exhibited distended, mucin-filled crypts in the ileum and excess mucus in the lumen of both the ileal and colonic regions (unpublished observation).

Data have been provided on the pattern of electrogenic Na+ absorption and Cl- secretion across three regions of the normal and CF neonatal murine intestine. In most CF mouse models, there is a very high rate of death due to intestinal problems during the first 3 days postbirth, with some strains of CF mice exhibiting a >90% mortality during the neonatal period (18). This high death rate correlates temporally with the high electrogenic amiloride-sensitive Na+ absorption (undoubtedly secondary to the elevated aldosterone levels) in the colonic epithelia and to a lesser extent in the ileum. Electrogenic Na+-nutrient cotransport (although less than normal) is also well developed at birth, especially in the proximal colon and ileum (and undoubtedly the jejunum) of the neonatal CF mouse. Therefore, the two regions most vulnerable to blockage and rupture exhibit a very high degree of electrogenic Na+ absorption at birth. This high rate of Na+ absorption, coupled with the complete inability to secrete Cl- in CF intestines, is predicted to increase net isotonic volume absorption in this region in CF and predispose the gut contents of these murine intestinal regions to dessication, resulting in impaction, blockage, and rupture.

In conclusion, the present study provides data on neonatal murine intestinal ion transport that demonstrate the feasibility of physiological studies on the neonatal murine intestine. The capacity to study the neonatal intestinal epithelium is useful to define phenotypic alterations in intestinal ion transport of various transgenic and knockout mouse models that may present as neonatal lethals and thus preclude study of these mutations in adult intestine. Also, gut disease affecting the neonate can be modeled and studied with these preparations. Ion transport across the normal neonatal murine intestine exhibits many similarities to the adult murine intestine, including Cl- secretion mediated by both an increase in cAMP (forskolin) and an elevation of intracellular Ca2+ (carbachol). In contrast to the adult murine distal colon (normal diet), the neonatal distal colon (normal and CF) exhibits a high rate of electrogenic amiloride-sensitive Na+ absorption. In addition, the neonatal murine distal colon, proximal colon, and ileum (normal and CF) exhibit significant Na+-nutrient cotransport. The importance of these Na+-nutrient cotransport pathways in the adult murine intestine has not been investigated in these intestinal regions. However, these transport pathways are significantly reduced in the proximal colon and ileum of the CF neonate, compared with the normal neonate, which may partially explain the lower rate of growth of these CF pups. It is speculated that the various types of electrogenic Na+ transport in the colon and ileum may be a variable that adds to the absence of cAMP or Ca2+-regulated Cl- secretion to predispose the neonatal CF mouse to intestinal obstruction and rupture.


    ACKNOWLEDGEMENTS

The helpful comments and support of Dr. R. C. Boucher are gratefully appreciated.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-42384.

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. §1734 solely to indicate this fact.

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 at Chapel Hill, Chapel Hill, NC 27599-7248 (E-mail: bgrubb{at}med.unc.edu).

Received 7 December 1998; accepted in final form 29 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(1):G167-G174
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