Lateral intercellular space volume as a determinant of CFTR-mediated anion secretion across small intestinal mucosa
Lara R. Gawenis,
Kathryn T. Boyle,
Bradley A. Palmer,
Nancy M. Walker, and
Lane L. Clarke
Dalton Cardiovascular Research Center and the Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
Submitted 5 November 2003
; accepted in final form 29 January 2004
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ABSTRACT
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Studies of full-thickness, small intestinal preparations have shown that maximal anion secretion [indexed by short-circuit current (Isc)] during intracellular cAMP (cAMPi) stimulation is transient and followed by a decline toward baseline. Declining Isc is preceded by decreases in transepithelial conductance (Gt), which in the small intestine reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMPi-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by Gt and electron microscopy) were investigated for an effect on the magnitude of cAMPi-stimulated anion secretion (assessed by the Isc and isotopic fluxes) across murine small intestine. In control studies, changes of Gt after cAMPi stimulation were associated with a morphological "collapse" of the LIS, which did not occur in intestine of CFTR-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution, or increased serosal hydrostatic pressure minimized reductions in Gt and increased the cAMPi-stimulated Isc response. Increased Isc primarily resulted from increased Cl secretion that was largely bumetanide sensitive. However, bumetanide-insensitive Isc was also increased, and similar increases occurred in the Na+-K+-2Cl cotransporter (NKCC1)-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.
bicarbonate; chloride; tight junction; Na+-K+-2Cl cotransporter; cystic fibrosis; mouse
VECTORIAL TRANSPORT OF ELECTROLYTES and water across epithelia involves coordination between transcellular processes of electrolyte transport and the paracellular shunt pathway. As recently reviewed by Spring (34), isotonic NaCl and water absorption has been most intensively studied and conforms to a modification of the three-compartment model originally proposed by Curran and MacIntosh (11). In the revised model, Na+ transport proteins of the apical membrane, in series with Na+-K+ ATPase at the basolateral membrane, provide a modest osmotic (Na+) gradient for water flux from lumen to cell and then to the lateral intercellular space (LIS) between the basolateral membranes of the epithelial cells. The slight hypertonicity of the LIS volume is sustained despite diffusion across the unstirred layer of the basement membrane and basal interstitial connective tissue and by a finite amount of NaCl back flux across the tight junction. Although less studied within this context, NaCl and water secretion across epithelia may also conform to the model. In the intestine, CFTR serves as the principal conductive pathway for stimulated secretion of chloride and bicarbonate anions (3, 10, 15, 27, 29). The anions available for secretion come from transport proteins of the basolateral membrane including the Na+-K+-2Cl cotransporter (NKCC1), anion exchangers (AE), and NaHCO3 cotransporters (NBCs), or by intracellular HCO3 generation through hydration of CO2 (6, 20, 30). These processes are capable of replenishing intracellular anions at rates that maintain a favorable driving force for apical efflux. By analogy with the absorptive three-compartment model, it would be predicted that a reverse osmotic gradient is established that allows water movement from a hypotonic LIS volume to a relatively hypertonic lumen.
Activation of anion secretion across the intestine by intracellular cAMP (cAMPi) stimulation has a distinct effect on the paracellular pathway. In the small bowel, as in other "leaky" epithelia, the transepithelial conductance (Gt) is largely a measure of the paracellular conductance rather than transcellular conductance (17, 26). The paracellular contribution to Gt in the small intestine is estimated to range in magnitude from 85 to 95% (26). The paracellular conductance reflects the contributions of the tight junction in series with the relatively more dynamic fluid compartment of the LIS. Earlier studies of another leaky epithelium, i.e., Necturus gallbladder, by Duffey et al. (14) demonstrated that cAMPi stimulation reduces the ionic permeability of the paracellular pathway. Although ultrastructural studies revealed reorientation of junctional complex strands by cAMPi stimulation (14), subsequent investigations have conclusively shown that the major effect of cAMPi stimulation in increasing resistance of the paracellular pathway is due to the so-called "collapse" of the LIS rather than a change in the resistance of the tight junctional complex (21, 37). Studies of intact intestinal preparations during the last 30 yr have shown that stimulation of anion secretion by increased cAMPi results in a delayed (2030 min) reduction in Gt (9, 19, 25). Restriction to paracellular ion movement has been verified by studies (19) showing a nearly equivalent reduction in the passive serosal-to-mucosal flux of Na+ across the intestinal epithelium. Ultrastructural studies (4) provide a morphological correlate in that stimulation of anion secretion causes a reduction in LIS volume as assessed by closer apposition of the basolateral membranes of intestinal epithelial cells. It is apparent from these studies that the fluid compartment of the LIS is taxed by acute induction of CFTR-mediated salt and water secretion in the intestine. A relatively finite volume in the LIS may limit the duration and perhaps magnitude of anion secretion by restricting anion availability or, through increased apposition of the basolateral membranes, set in motion signaling events that affect basolateral transporter activity.
Studies of electrogenic anion secretion across the intestine during acute stimulation of cAMPi activity often show that the rate of secretion as indexed by short-circuit current (Isc) has a biphasic pattern (18, 25). After a rapid increase to a maximal rate, the Isc declines substantially over several minutes to a rate that is elevated above baseline. Because Gt declines within the same time frame, it is possible that the collapse of the LIS is a major physical factor resulting in the subsequent decline in Isc. Therefore, as an extension to earlier studies showing cAMPi regulation of LIS permeability, we hypothesized that the decrease in Gt during acute cAMPi stimulation reflects changes in LIS volume that limit the magnitude of transepithelial anion secretion. To test this hypothesis, we investigated whether induced alterations of the LIS volume (as indexed by Gt and electron microscopy) would result in predictable changes in the magnitude of the cAMPi-stimulated anion secretion across murine jejunum.
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MATERIALS AND METHODS
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Animals.
The experiments in this study used wild-type (WT) mice and mice with gene-targeted disruption of CFTR (cftrtm1Unc) or NKCC1 (locus Slc12a2) (16, 33). All mice were maintained on standard laboratory chow (Formulab 5008 Rodent Chow; Ralston Purina, St. Louis, MO). The mice were genotyped between 3 and 6 wk of age as previously reported and identified as WT, CFTR knockout [CFTR()], or NKCC1 knockout [NKCC1()] (7). To prevent intestinal impaction in the CF mice, an oral osmotic laxative (polyethylene glycol; mol wt 3,350) was provided ad libitum in the drinking water of the CF mice and their WT littermates (7); all other mice were provided with tap water. Mice ages 24 mo were used in the experiments. Before experimentation, the mice were fasted overnight and drinking water was provided ad libitum. All experiments were approved by the University of Missouri Animal Care and Use Committee.
Bioelectric measurements.
Excised proximal duodenum or midjejunum (antimesenteric side) was mounted in standard Ussing chambers (0.238-cm2 surface area) as previously described (9, 19). For some experiments, the smooth muscle layer was removed by blunt dissection (muscle stripped). Mucosal and serosal surfaces of the intestinal segments were bathed independently with warmed (37°C), oxygenated Krebs bicarbonate Ringer solution of the following composition (in mM): 140 Na, 119.8 Cl, 25 HCO3, 5.2 K, 2.4 HPO4, 0.4 H2PO4, 1.2 Mg, and 1.2 Ca, pH 7.4. Glucose (10 mM) was added to the serosal bath, whereas in the mucosal bath, mannitol (10 mM) was substituted for glucose to avoid an inward current due to Na+-coupled glucose cotransport (8). To minimize the contribution of endogenous prostaglandins and neural activity, all tissues were treated with indomethacin (1 µM) and TTX (0.1 µM) (5, 9, 31). cAMPi was stimulated by the addition of forskolin (10 µM) and IBMX (100 µM) to both the mucosal and serosal baths. For experiments using hypertonicity, 250 mM polyethelene glycol (mol wt 200) was added to the bathing medium. Transepithelial Isc (reported as µA/cm2 and µeq·cm2·h) was measured using an automatic voltage clamp (model VCC-600; Physiologic Instruments, San Diego, CA). Gt (mS/cm2) was calculated by measuring the current deflection after the application of a 5-mV pulse and applying Ohm's law. All experiments were performed under short-circuited conditions (serosal bath as ground).
Radioisotopic fluxes.
Unidirectional mucosal-to-serosal (Jms), serosal-to-mucosal (Jsm), and net fluxes (Jnet = Jms Jsm) of 36Cl were estimated from two 30-min flux periods, as described previously (19). The first 30-min flux period served as a control (basal). This was followed by treatment with forskolin/IBMX that was added 30-min before a second 30-min flux period (cAMPi) was performed for comparison. Jejunal tissues from each mouse were paired by Gt (within 10%) for the calculation of Jnet. For net fluxes, a positive sign indicates net Cl absorption (mucosal to serosal) and a negative sign indicated net Cl secretion (serosal to mucosal).
Transmission electron microscopy.
Small intestine sections were mounted in Ussing chambers as described above. After an experimental period, intestinal segments were bathed in a fixative containing 2.5% glutaraldehyde, 2.0% paraformaldehyde and (in mM) 70 NaCl, 30 Na HEPES, and 2 CaCl2. Two-millimeter sections of the fixed specimens were fixed in 1% osmium tetroxide and 1% uranyl acetate, dehydrated with ethanol and infiltrated with epoxy resin. Thin sections (80 nm) were cut and placed on 200-mesh copper grids before staining with uranyl acetate and lead citrate. Sections were viewed using a JEOL 1200EX transmission electron microscope at 80 kV accelerating voltage.
Statistics.
A two-tailed Student's t-test assuming equal variances was used to compare data between two treatment groups. For comparisons among multiple groups, a one way-ANOVA with a post hoc Tukey's t-test was used. A probability value of P < 0.05 was considered statistically significant. All values are reported as means ± SE.
Materials.
36Cl was obtained from PerkinElmer (New England Nuclear; Boston MA). All other reagents were obtained from either Sigma (St. Louis, MO) or Fisher Scientific (Springfield, NJ).
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RESULTS
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Stimulation of cAMPi in full-thickness small intestinal preparations not only induces transepithelial secretion but also significantly decreases the Gt (9, 18, 19, 25). As shown in Fig. 1A, the addition of a cocktail of 10 µM forskolin and 100 µM IBMX (F/I) leads to a rapid increase in Isc, which attains a peak value within 10 min. As the Isc reaches a maximum, Gt begins to decrease and this change precedes a decline in the Isc. Both the Isc and Gt reach a stable plateau
20 min after F/I addition. In a leaky epithelium like the small intestine, changes in Gt largely reflect changes in LIS volume of the paracellular pathway (4, 21, 25, 37). Transmission electron microscopy of intact intestinal epithelium was used to verify that changes in the magnitude of the Gt correlate with morphological changes in the LIS. Under basal conditions when Gt is highest (Fig. 1A), the LIS is relatively open in murine jejunum (Fig. 2A); but 20 min after F/I treatment when Gt is decreased, the LIS is collapsed as indicated by close apposition of the basolateral cell membranes (Fig. 2B). The reduction in Gt and collapse of the LIS after stimulation of cAMPi is dependent on the induction of CFTR-mediated anion secretion, because Gt is not altered (Fig. 1B) and the LIS remains open (Fig. 2C) after F/I treatment of jejuna from CFTR knockout mice. Thus, in the WT small intestine, decreased Gt after stimulation of cAMPi indicates a shift of fluid volume out of the LIS during CFTR-mediated anion secretion. The fact that decreased Gt precedes the decline in Isc after F/I treatment suggests that the LIS volume may be a physical determinant with regard to the availability of electrolytes and water of anion secretory capacity of the small intestinal mucosa. The dynamic nature of this relationship is shown by the experiments (depicted in Fig. 1C) in which bumetanide inhibition of stimulated Cl secretion not only decreases the Isc, but also results in the recovery of Gt toward its baseline value. The magnitude of the LIS volume (as indexed by Gt) under basal, nonstimulated conditions is also an accurate predictor of the magnitude of a subsequent cAMPi-induced anion secretory response. As shown by Fig. 1D, a plot of the maximal cAMPi-stimulated
Isc as a function of the basal Gt in WT small intestine shows a significant positive correlation, i.e., intestinal preparations with greater basal Gt exhibit an increased maximal
Isc after the addition of F/I. The above observations are consistent with the hypothesis that changes in LIS volume (as indexed by Gt) lead to predictable changes in the magnitude and duration of transepithelial anion secretion. To test this hypothesis, several experimental approaches were used to manipulate the magnitude of the LIS before induction of cAMPi-stimulated anion secretion.

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Fig. 1. A: time course of short-circuit current (Isc) and transepithelial conductance (Gt) responses of wild-type (WT) murine jejunum to treatment with a cocktail of 10 µM forskolin and 100 µM IBMX (cAMP). Note that the decline in Gt after forskolin/IBMX treatment precedes the decline in the Isc from its maximal value (n = 6). B: time course of Isc and Gt responses of CFTR knockout murine jejuna to forskolin/IBMX treatment (n = 6). C: time course of Isc and Gt responses of WT jejuna to sequential treatments with forskolin/IBMX followed by 50 µM bumetanide (Bumet) (n = 6). D: linear relationship between the basal Gt and the maximal Isc in response to forskolin/IBMX treatment in WT jejuna (n = 22 jejuna; R = 0.84, P < 0.05).
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Fig. 2. Ultrastructural studies of the lateral intercellular spaces (LIS) of jejunal mucosa. A: untreated WT murine jejunum. B: WT murine jejunum 20 min after treatment with a cocktail of 10 µM forskolin and 100 µM IBMX. C: CFTR knockout murine jejunum 20 min after treatment with forskolin/IBMX. D: WT murine jejunum after sequential treatment with + 250 mosM PEG in the serosal bath and forskolin/IBMX. LIS indicated by arrows. Transmission electron micrographs are representative of sections taken from 4 WT and 3 CFTR knockout mice. Magnification, x4,000.
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Previously, we and others (12, 18) have observed that anisosmotic bathing solutions alter the Gt in the small intestine. Exposure of intact jejunum to a hypertonic solution (isotonic + 250 mosM) in the luminal bath decreases the Gt to levels similar to that attained by cAMPi stimulation (18) and significantly limits the subsequent Isc response to increased cAMPi (see Fig. 3C, left). Interestingly, exposure of intact jejunum to a hypertonic solution in the serosal (basolateral) bath increases Gt and the magnitude and duration of the Isc response to F/I addition. As shown in Fig. 3A, serosal hypertonicity (isotonic +250 mosM) prevents the decrease in Gt that normally occurs during cAMPi stimulation of intact jejunum, presumably by inducing sustained fluid movement through the LIS in a mucosal-to-serosal direction (37). Ultrastructural studies confirm that LIS volume during exposure to serosal hypertonicity is maintained after 20-min stimulation with F/I (Fig. 2D). Most importantly, exposure to serosal hypertonicity also resulted in a greater maximal
Isc in response to cAMPi stimulation that was sustained over 20 min, in contrast to the Isc response in the jejunum bathed in isotonic medium (Fig. 3, B and C, right).
Studies of murine duodenum indicate that dissection of the outer intestinal musculature from the mucosa (i.e., muscle stripping) minimizes cAMPi-induced reductions in Gt (10). To evaluate our present hypothesis, bioelectric parameters during basal and cAMP-stimulated conditions were compared between intact and muscle-stripped intestine in both murine duodenum (Fig. 4A) and jejunum (Fig. 4B). Under basal conditions, muscle-stripped intestine had a significantly greater Gt than intact intestine, but the Isc was similar between the two groups. After cAMPi stimulation, Gt decreased very little (
10%) in the muscle-stripped intestine, and the Isc increased rapidly to a maximal value that was sustained over the next 15 min. In contrast, in intact intestine, Gt decreased by >50% after F/I treatment, and the maximal change in Isc was significantly less (and decreased rapidly) compared with the muscle-stripped preparations (cumulative Isc data shown). Subsequent treatment with bumetanide decreased the Isc in both groups but substantially increased Gt only in the intact intestine. As a test of tissue viability in these studies, the Na+-coupled glucose current as indexed by the Isc response 10 min after the mucosal addition of glucose (10 mM mucosal) was measured at the end of each experiment. No differences in viability were detected between intact and muscle-stripped tissue preparations [intact = 87.5 ± 13.3 µA/cm2 (range = 55.0 to 131) vs. muscle-stripped = 127.6 ± 17.5 µA/cm2 (range 76.5 to 168.1); n = 6; P not significant].
Comparison of intact vs. muscle-stripped intestine carries the advantage that the studies can be performed with balanced salt solutions on each side of the preparation, making possible isotopic flux measurements of the two groups. Therefore, to determine whether the enhanced cAMPi-induced Isc in the muscle-stripped intestine was due to increased Cl secretion, unidirectional and net Cl fluxes across murine jejunum were measured during both basal and cAMPi-stimulated conditions. As shown in Table 1, under basal conditions, muscle stripping increased the Gt and, surprisingly, also increased net Cl absorption as a result of an increased absorptive (mucosal-to-serosal) unidirectional flux of Cl. Subsequent cAMPi stimulation with F/I resulted in net Cl secretion and an increased Isc (converted to flux units to facilitate comparison with JnetCl) in both intact and muscle-stripped preparations. However, net Cl secretion was significantly greater in the muscle-stripped jejunum (+3.3 µeq·cm2·h vs. intact), which compares favorably with a greater increase in Isc (+3.8 µeq·cm2·h vs. intact). Thus a major portion of the enhanced Isc response to cAMPi stimulation in the muscle-stripped preparation is electrogenic Cl secretion. A comparison of changes in the unidirectional Cl fluxes between the two groups was not possible, because the passive component of the transepithelial Cl flux in the intact jejunum is substantially reduced as a consequence of the decrease in the paracellular pathway after F/I treatment (as reflected by the significantly decreased Gt in these preparations).
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Table 1. Unidirectional and net 36CI fluxes, short-circuit current, and transepithelial conductance in intact and muscle-stripped jejuna
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As shown above, application of a sustained hydraulic pressure gradient across the murine intestine by anisosmotic conditions prevented collapse of the LIS (as indexed by the Gt) and increased the magnitude of the Isc response to cAMPi stimulation. Earlier studies by Kottra et al. (21) using Necturus gallbladder have shown that small hydrostatic pressure gradients continuously distend the LIS. Therefore, as a third means to manipulate the LIS volume in the intestinal preparations, hydraulic pressure was directly applied to the serosal side of the jejunum by increasing hydrostatic pressure.
As shown in Fig. 5A, the Gt of jejuna bathed with equal hydrostatic pressures in the serosal and mucosal baths (balanced) decreased during the time period before F/I stimulation, whereas the Gt was unchanged in jejuna exposed to +2.9 mmHg and significantly increased in jejuna with +3.7 mmHg increase in the serosal hydrostatic pressure. As shown in Fig. 5B, the change in Gt resulting from the addition of +3.7 mmHg hydrostatic pressure in the serosal bath was sustained during F/I stimulation and, as shown in Fig. 5, C and D, this change was associated with a significantly greater cAMP-stimulated Isc response. Interestingly, the small increases in serosal hydrostatic pressure were not sufficient to completely prevent the collapse of the LIS during F/I stimulation. In all three groups, Gt declined during cAMP stimulation (balanced
Gt = 8.0 ± 4.8; +2.9 mmHg
Gt = 17.5 ± 0.9; +3.9 mmHg
Gt = 13.2 ± 4.8 mS/cm2) and preceded a decline in Isc after peak stimulation (see Fig. 5C).
As shown by the time course studies (Figs. 35) and as depicted graphically in Fig. 6 for the serosal hypertonicity and muscle-stripping experiments, manipulations to increase LIS volume resulted in a significantly increased bumetanide-sensitive Isc. Thus Cl secretion supported by the activity of the basolateral cotransporter NKCC1 is significantly increased when LIS volume is sustained during cAMPi stimulation of the murine small intestine. However, as also shown in Fig. 6, the bumetanide-insensitive (or residual) Isc is also increased by manipulation of the LIS volume. This observation suggests that sustaining LIS patency also increases CFTR-dependent anion secretion that is supported through anion uptake by other transport proteins at the basolateral membrane, e.g., AE2 and NBCs (20, 36). Previous studies (36) of duodenal anion transport in NKCC1 knockout mice have shown that an alternative SITS-sensitive Cl uptake pathway at the basolateral membrane (possibly the AE2 anion exchanger) supports a finite rate of CFTR-mediated Cl secretion. Chloride secretion mediated by this mechanism in the murine duodenum occurs in parallel with an approximately equivalent proportion of CFTR-mediated bicarbonate secretion that is supported by basolateral NaHCO3 cotranporters and hydration of CO2 (1, 2, 36). To investigate the effect of LIS volume on non-NKCC1 uptake pathways that are activated during cAMPi-stimulated anion secretion, we assessed the linear relationship between the basal Gt and the SITS-sensitive Isc after cAMPi stimulation using duodena from NKCC1 knockout mice. Addition of 1 mM SITS to the serosal bath after F/I treatment has been previously shown to inhibit the alternative Cl uptake mechanism in murine duodenum and likely has inhibitory effects on basolateral NaHCO3 cotransport pathways (22, 36). As shown in Fig. 7, the SITS-sensitive
Isc demonstrated a significant positive correlation with the Gt, indicating that other pathways of anion uptake that support CFTR-mediated anion secretion (e.g., AE2, NBCs) are also affected by the existing LIS volume in intestinal epithelia.
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DISCUSSION
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Transcellular secretion of anions during stimulation of cAMPi involves the interplay of several epithelial cell processes including an apical conductive pathway (CFTR), basolateral transport proteins, signaling events, and enzymatic activity. The interaction of these processes has been intensively studied, oftentimes in relative isolation, but another level of complexity is introduced when epithelial transport is considered within the context of an intact structure like the intestinal wall. In studies (25) confirming observations made by Powell 30 yr ago, it was shown that the rate of transepithelial anion secretion during acute cAMP stimulation is biphasic in that maximal secretion is transient and followed by a rapid decline. Preceding the decline in anion secretion is a significant decrease in tissue conductance, which, in the case of the small intestine, indicates a reduction in paracellular conductance resulting from a collapse of the LIS (4, 18). It was hypothesized that the inability of intact intestinal epithelium to sustain maximal rates of cAMPi-stimulated anion secretion may be secondary to a limitation in the availability of anions and water for uptake from the LIS volume. Various experimental conditions (muscle-stripping, hydrostatic and hydraulic pressure gradients) intended to alter the LIS volume (as indexed by Gt) resulted in predictable changes in the magnitude and duration of cAMPi-stimulated anion secretion. The results support the concept that collapse of the LIS limits the duration of maximal rates of anion secretion during cAMPi stimulation of intact intestine. Ultrastructural studies confirmed a correlation between LIS patency and changes in Gt.
The major component of the cAMPi-stimulated Isc affected by manipulation of LIS volume was electrogenic chloride secretion. Isotopic flux studies demonstrated that an increase in cAMPi-stimulated Isc resulting from muscle-stripping was closely associated with an increase in net Cl secretion. Furthermore, it was consistently found that the magnitude of bumetanide-sensitive Isc was increased by all manipulations designed to increase LIS volume. The latter observation indicated that the activity of NKCC1 for basolateral uptake of Cl was sustained when the LIS remained patent during CFTR-mediated secretion. A number of factors are involved in increased activity of NKCC1 during stimulated secretion, including elevated cAMPi, cell volume reduction, decreased intracellular Cl activity, alteration in the cytoskeleton, and changes in the number of available transporters (24). These alterations in the cellular environment stimulate NKCC1 both to increase the basolateral uptake of Cl in support of secretion and to recover cell volume and Cl content toward homeostatic values. Although the activity of NKCC1 is not generally considered to be the rate-limiting step in CFTR-mediated Cl secretion (24), the results of the present study suggest that LIS volume can significantly affect the magnitude and duration of cAMPi-stimulated Cl secretion either by restricting ion availability to NKCC1 or by downregulating the activity of the cotransporter.
The dependency of cAMPi-stimulated anion secretion on LIS patency is not limited to NKCC1-facilitated transepithelial chloride secretion. It has been recently reported (36) that basolateral uptake of Cl to support transepithelial secretion can be accomplished in the complete absence of NKCC1 by SITS-sensitive transport, possibly AE2 activity. Additionally, a component of the Isc response to cAMPi stimulation in murine small intestine represents CFTR-mediated electrogenic HCO3 secretion (10, 36). Sources of HCO3 for secretion across the intestine include the activity of NaHCO3 cotransporters [e.g., the electrogenic NBC1 (2)] located at the basolateral membrane, as well as spontaneous and catalyzed hydration of CO2 (1, 10). Similar to NKCC1, activity of the electrogenic NBC1 is increased during stimulated anion secretion possibly due to the favorable electrical gradient that develops upon activation of the CFTR anion conductance (13, 23, 32). In the present study, several observations were consistent with the proposal that non-NKCC1 pathways of anion uptake were also affected by LIS patency. First, in WT intestine, the bumetanide-insensitive Isc was significantly increased under conditions that maintained the Gt during cAMPi stimulation. Second, although our studies were limited in number, examination of ion transport across the NKCC1 knockout intestine suggested a similar relationship between the Isc and Gt. In the case of the SITS-sensitive component of the cAMPi-stimulated Isc, a highly significant correlation existed with the Gt. Although SITS at the concentration used in these experiments may affect NaHCO3 cotransport activity in addition to the inhibition of AE2 (28), it seems clear that the activity of transport proteins that support cAMPi-stimulated anion secretion in the absence of NKCC1 are also affected by the LIS patency.
Several physiological implications follow from the observation that the LIS volume is a determinant of the magnitude of stimulated anion and thus fluid secretion. First, the magnitude of intestinal anion secretion would be partially dependent on the changes in LIS volume resulting from alterations in the submucosal Starling forces. For example, increased hydrostatic pressure resulting from increased perfusion pressure might maintain LIS volume and support intestinal secretion, whereas reductions in perfusion would have the opposite effect. These changes are consistent with the physiology of enterosystemic fluid movement during digestion and the effects of sympathetic stimulation (stress response). Second, the dynamics of the LIS may facilitate preservation of systemic fluid balance during changes in luminal osmolarity. We and others (12, 18) have shown that exposure of intact murine jejunum to hypertonic luminal solutions decreases Gt, which significantly reduces transepithelial anion secretion in response to cAMPi stimulation (Fig. 3C). Thus active secretion is diminished in the presence of osmotically induced secretion. In contrast, exposure of the bowel to serosal hypertonicity sustains LIS volume and high rates of anion secretion. In this case, excessive fluid absorption induced by serosal hypertonicity is counterbalanced by facilitation of anion secretion, thereby acting to preserve systemic fluid balance. Although the degree of hyperosmolarity applied to the serosal compartment in these experiments is unlikely to be encountered in most physiological conditions, recent studies (35) of active Na+ absorption in descending colon demonstrate a hypertonic absorbate (estimated at 491 mM) in the pericryptal space of the submucosa. If the magnitude of the paracellular conductance is also an accurate predictor of passive fluid convection in response to osmotic gradients across the epithelium, then changes in the LIS (or the anatomical arrangement of basolateral cell membranes and tissues bordering the LIS) are consistent with the action of a one-way valve. Exposure to serosal hypertonicity increases Gt, which facilitates absorptive fluid convection, whereas exposure to mucosal hypertonicity decreases Gt and limits secretory fluid convection. In the latter case, it can be seen from the preceding discussion that both passive and active secretions are limited during increased luminal osmolarity, thereby helping to maintain systemic volume homeostasis when hypertonic content is present within the intestine. Third, the effect of LIS volume on anion secretion should also be considered in states in which intestinal electrolyte absorption is reduced. Recent studies (19) of mice with gene-targeted deletion of the Na+/H+ exchanger NHE3, a major protein involved in intestinal Na+ absorption, demonstrate both a reduced intestinal Gt under basal conditions and a limited capacity for anion secretion in response to cAMPi stimulation. Finally, the studies of muscle-stripped intestine suggest that the integrity and perhaps tone of the outer intestinal musculature are important to the physiological relationship between LIS volume and anion secretion. Overall, the present evidence suggests that LIS patency in the intact intestinal wall is an important determinant of mucosal electrolyte and water secretion. The role of the LIS compartment therefore should be considered when evaluating the physiological context of cellular processes of electrolyte transport.
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GRANTS
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48816 (to L. L. Clarke).
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge the expert technical assistance of Cheryl Jensen at the University of Missouri Electron Microscopy Core.
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FOOTNOTES
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Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, 134 Research Park Dr., Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: clarkel{at}missouri.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.
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