NSP4 elicits age-dependent diarrhea and Ca2+mediated Iminus influx into intestinal crypts of CF mice

Andrew P. Morris1, Jason K. Scott1, Judith M. Ball2, Carl Q.-Y. Zeng2, Wanda K. O'Neal3, and Mary K. Estes2

1 Departments of Integrative Biology and Internal Medicine-Gastroenterology, University of Texas at Houston Health Science Center, and 2 Divisions of Molecular Virology and Medicine-Gastroenterology and 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4114-135 peptide, can overcome these gastrointestinal complications in CF (CFTRm3Bay null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR+/+), heterozygous (CFTR+/-), or homozygous (CFTR-/-) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca2+ mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I- influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR-/- background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca2+ or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca2+-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4114-135 to counteract chronic constipation/obstructive bowel syndrome in CF patients.

cystic fibrosis transmembrane conductance regulator; halide permeability; NSP4 enterotoxin; rotavirus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOST COMMONLY REPORTED gastrointestinal manifestation in cystic fibrosis (CF) is malabsorption due to pancreatic insufficiency, caused by occlusion of the pancreatic ducts by abnormally viscous mucus secretions. This alteration in intraluminal fluid and mucus content extends to other solid organs and hollow viscera of the gut, creating the clinical conditions of meconium ileus at birth and distal intestinal obstruction syndrome, chronic constipation with acquired megacolon, and rectal prolapse in older individuals (1).

Underlying these gastrointestinal complications is the basic defect in CF, which is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR; Ref. 43). The most commonly reported mutation in CF, accounting for the genetic defect in 60-70% of the chromosomes of CF individuals, is a three-base-pair deletion that removes phenylalanine at position 508 (Delta F508) of the CFTR protein (54). CF patients homozygous for this mutation exhibit the severe clinical phenotype of lung disease, pancreatic insufficiency, and predisposition to gastrointestinal obstruction (48). Electrophysiological analysis of the gut mucosa isolated from these individuals demonstrates a lack of cAMP-dependent fluid secretion (6, 37, 53). Reduced fluid transport, which is the primary cellular defect of CF epithelia, is believed to significantly enhance the pathology of mucus plugging in the gut.

Over the past six years, mouse models for CF that duplicate the pathophysiological phenotype of CF in the human intestine have been produced with the use of gene-targeting techniques. Transgenic CFTR-deficient animals fail to exhibit CFTR-mediated fluid secretion and present with gastrointestinal disease (15). In this study, we utilized the CFTRm3Bay null mutation mouse as a model for CF to investigate whether infectious rotavirus or the novel rotaviral enterotoxin NSP4 (3) can cause diarrhea. These mice fail to express functional CFTR protein because of multiple stop codons engineered within exon 3 of the murine CFTR genome (23).

Rotaviruses are the leading cause of severe gastroenteritis in infants and young animals (25). We have recently shown that NSP4, a rotavirus nonstructural protein (16), can cause diarrhea in young mice (3). Furthermore, electrophysiological analyses of intact intestinal mucosa from mice revealed that NSP4 mobilizes Ca2+ to mimic the secretory effects of the cholinergic agonist carbachol (CCh) in potentiating cAMP-dependent fluid secretion (3). We therefore asked whether NSP4 could evoke a diarrheal response in CFTR-depleted mice that exhibit no functional cAMP-dependent secretory pathway. Here, we demonstrate that NSP4 injected intraperitoneally into CFTR-deficient (CFTR-/-) mouse pups induces an age-dependent diarrhea. Hence, CFTR-mediated changes in intestinal fluid transport were not directly involved in NSP4-elicited diarrhea. However, NSP4 was found to mobilize Ca2+ in the crypt epithelia of both small and large intestine from wild-type (CFTR+/+) as well as CFTR-/- pup and adult mice. These ex vivo data correlated with our in vitro findings in human gastrointestinal cell lines, in which NSP4 was shown to mobilize intracellular Ca2+ concentration ([Ca2+]i) via phospholipase C (PLC) activation and inositol 1,4,5-trisphosphate (IP3) production (13). To investigate in greater detail the relationship between NSP4-induced epithelial cell Ca2+ mobilization and our previously reported age-dependent effects of NSP4 on murine intestinal mucosa Cl- secretory current generation (3), we measured plasma membrane halide permeability changes ex vivo within the epithelial cells of isolated distal colon crypts. We found that NSP4 elicited age-dependent I- influx into mouse pup crypts, inhibited by either the removal of bath Ca2+ or the transport inhibitor DIDS. The fact that this Ca2+-dependent plasma membrane halide permeability pathway was not CFTR may provide a possible explanation as to why CFTR-/- mouse pups exposed to NSP4 develop age-dependent diarrhea.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Fura 2-AM and 6-methyoxy-N-(3-sulfopropyl) quinolinum (SPQ) were purchased from Molecular Probes (Eugene, OR). All other reagents, including DIDS, were purchased from Sigma (St. Louis, MO). NSP4 protein was fast-performance liquid chromatography- and affinity-purified from Sf9 insect cells infected with a recombinant baculovirus pAC461-G10 expressing simian rotavirus gene 10 (encoding NSP4), as described previously (13). The NSP4114-135 peptide was synthesized at the University of Pittsburgh protein core laboratory and characterized as previously described (3).

Experimentally induced rotaviral infection or administration of NSP4 protein and NSP4114-135 peptide to mouse pups. The simian rotavirus strain SA11 clone 3 (18) was used to infect neonatal CD-1 or CFTR-/- (23) mouse pups between the ages of 6 and 8 days. The genotype of the CFTR-/- mice was determined by polyacrylamide gel analysis of PCR products generated from DNA extracted from the tails of 1- to 2-day-old mice with the use of primers, as described previously (23). Either 10 or 20 diarrheal dose 50 of SA11 was administered in 50 µl of medium 199 by intragastric gavage. Alternatively, purified NSP4 protein or NSP4114-135 was administered to C57B CFTR-/-/CFTR+/+ or CD-1 CFTR+/+ mouse pups in a final volume of 50 µl of PBS by intraperitoneal injection. The doses used were 0.5 nmol of NSP4 protein or 100 nmol of NSP4114-135 peptide because these doses were shown to be effective at inducing diarrhea in CD-1 CFTR+/+ mouse pups (3).

Diarrheal activity measurements. To determine the presence of diarrhea following protein or peptide treatment, each mouse pup was examined every 1-2 h for the first 8 h and at 24 h after inoculation by gently pressing the abdomen. After virus inoculation, mouse pups were monitored twice a day for 4 days. Diarrhea was noted and scored from 1 to 4, with a score of 1 reflecting loose yellow stool and a score of 4 indicating completely liquid stool. A score of 2 (mucous with liquid stool, some loose but solid stool) and above was considered diarrhea. The scoring was performed on coded animals by a single person.

Crypt isolation and dye loading. Epithelial crypts from CFTR-/- mice (aged 8-12 days) were isolated from 3-cm segments of the mid to distal small intestine, 6 cm proximal to Bauhin's valve, and from the distal colon. After euthanasia by an overdose of ether and cervical dislocation, the entire small intestine and colon from the mouse were removed and flushed with ice-cold physiological saline. Individual intestinal segments were then mounted onto Perspex paddles and were immersed in Ca2+-free standard Krebs buffered saline (in mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4, 1.8 Na2HPO4, 10 glucose, and 10 EDTA), and continuously gassed with 5% CO2-95% O2 at 37°C for 10-20 min. The crypts were then separated from the overlying mucosa by mechanical vibration for 30 s into ice-cold KCl HEPES saline (in mmol/l: 100 potassium gluconate, 20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 5 sodium pyruvate, and 0.1% BSA, pH 7.4), resembling the intracellular medium. Suspended crypts were then deposited (1,200 rpm for 1 min) onto poly-L-lysine-coated microscope coverslips (0 oz) with the use of a Shandon Cytopsin cell preparation system (13). The coverslips with crypts were then attached with vacuum grease to the base of customized perfusion wells and loaded in the dark with either Ca2+-sensing or Cl--sensing dye.

Fura 2-AM loading. Isolated crypts were incubated with 10 µM fura 2-AM at room temperature for 15-20 min. The coverslips were then mounted on an inverted Nikon microscope and superfused with standard HEPES-buffered extracellular saline (in mmol/l: 140 NaCl, 4.7 KCl, 1.13 MgCl2, 10 HEPES, 10 glucose, and 1 CaCl2, pH 7.4) for 5 min before imaging.

SPQ loading. Isolated crypts were loaded with SPQ at room temperature by a 6-min exposure to hyposmotic (80 mosM Cl- or NO-3) HEPES-buffered extracellular saline containing 5 mM SPQ. After loading, the crypts were allowed to recover in isosmotic (140 mosM Cl- or NO-3) HEPES-buffered extracellular saline for 10-15 min. This period was used when performing anion channel inhibitor studies to preload the crypts so that the reported spectral interference of 0.5 mM DIDS with SPQ fluorescence was minimized (61). DIDS binds irreversibly to cellular membranes; therefore, crypts were loaded and the bathing saline was changed before experimentation. The solution temperature was maintained at 37°C throughout these studies by prewarming the extracellular solutions and by water-jacketing the oil-immersion lens of the inverted microscope.

NSP4/NSP4114-135 addition to isolated crypts. Small (100 µl) volumes of either the NSP4 protein or NSP4114-135 peptide were superfused onto the isolated crypts during regular bath flow by N2 pressure injection with the use of a Picospritzer. Low-resistance glass pipettes were filled with either compound dissolved in HEPES-buffered extracellular saline, and the tip was maneuvered close to the isolated crypt. Peptide or protein was released directly into the vicinity of the crypt for 40 s before being washed away by the bath flow.

[Ca2+]i imaging. All experiments were carried out with the aid of a high-resolution camera imaging system as described previously (13). In brief, light emitted from fura 2-loaded cells at 510 nm was captured by an intensified video camera after exposure to both 340- and 380-nm excitation light. The camera signal was then digitally encoded and processed with image analysis software (IMAGE1/FL, Universal Imaging, Media, PA). The background-subtracted images were ratioed on a pixel-by-pixel basis to yield a bitmap field. Calibration of the fura 2 dye fluorescence was carried out with the use of the ionophore ionomycin under Ca2+-free and Ca2+-saturating conditions as described previously, and the [Ca2+]i was calculated according to the Grynkiewicz equation (21). The [Ca2+]i values of individual field pixels obtained by this procedure were color coded and displayed on an RGB monitor before being stored on the hard disk. Six collection areas were chosen along the longitudinal axis (base to neck) of the crypt for spatial and time-dependent analysis of [Ca2+]i. The averaged ratio signal obtained from each of the six areas was digitally saved as a log file. The collected values from the six areas imaged within a single experiment were averaged together to give an experimental observation of one (n = 1). Values obtained from similarly placed collection areas along the longitudinal axis of different crypts were also averaged to see if any spatial localization to the Ca2+ signal existed. All protocols were completed within 1 h after crypt isolation.

Intracellular I- imaging. The same high-resolution camera imaging system described above was utilized for these studies. Excitation for SPQ fluorescence was provided by a barrier filter centered at 365 ± 10 nm, reflected into the microscope objective by a 400-nm dichroic mirror. A neutral density filter (1.5 OD) was included in the light path to minimize photobleaching. The fluorescence emission from the SPQ-loaded cells passed through a 450 ± 25-nm barrier filter before being detected by the intensifier/camera. Fluorescent images were acquired at 0.8-s intervals and averaged over eight frames to yield a 540 × 480 field of mean pixel intensities. Six collection areas were then chosen within the longitudinal axis (base to midregions) of the crypt for spatial and time-dependent analysis of cellular SPQ fluorescence. SPQ does not exhibit excitation or emission shifts on anion interaction. Thus ratiometric imaging is not possible, and dye fluorescence is affected by volume changes within the cell (58). Both cAMP- and Ca2+-mobilizing agonists have been reported to induce shrinkage without compensatory volume recovery in the lower to midregion of rat distal colonic crypts over a 5- to 30-min time course (11). To determine the extent of this effect over the time period of SPQ quenching (1-3 min), colonic crypts isolated from the distal colon of the mouse were loaded with the AM form of calcein, a dye inert to Ca2+ and pH changes in cells within the physiological range (8, 9). Cell volume measured over the first 30 s of agonist addition to the bath did not change appreciably (<2%) within the lower/midcrypt region (data not shown). These regions were utilized for all studies. Furthermore, to minimize possible volume effects on dye fluorescence, we chose an experimental design in which SPQ fluorescence quench by halide influx was employed rather than dequenching following halide efflux. This ensured that agonist-induced cell shrinkage, if present, would act to decrease rather than to potentiate changes in plasma membrane halide permeability. Thus our reported values of SPQ quench may modestly underestimate agonist-evoked halide influx. The collected values from the six areas imaged within a single experiment were averaged together to give an experimental observation of one (n = 1).

Calculation of Stern-Volmer constants for halide quenching of SPQ. SPQ fluorescence is quenched collisionally by halide anions with different potency. Nonphysiological anions such as thiocyanate (SCN-), Br-, F-, and I- quench strongly in free solution with an efficacy greater than Cl- (Cl- < Br- < SCN- < I-), whereas NO-3 does not quench at all (27). The Stern-Volmer equation that describes this interaction is Fo/Fanion = 1 + Kq[anion], where Fo is the fluorescence in the absence of anion, Fanion is the fluorescence in the presence of a given anion concentration, Kq (slope) is the Stern-Volmer quenching constant (in M-1), and [anion] is the concentration of halide utilized (27). Stern-Volmer quenching constants for Cl-, Br-, and I- anions were calculated from linear curves obtained over a concentration range of 0-150 mM halide (see RESULTS). The near-linear Stern-Volmer quench curve for I- (Fig. 1) and the fact that plasma membrane I- permeability changes occur largely through conductive pathways (see DISCUSSION and Refs. 36 and 63) led us to choose this anion to quantify agonist effects in isolated crypts.


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Fig. 1.   Stern-Volmer plot for quenching of 6-methoxy-N-(3-sulfopropyl)quinolinum (SPQ) fluorescence by Cl-, Br-, and I-. Isolated crypts from distal colon of wild-type cystic fibrosis transmembrane conductance regulator (CFTR+/+) and heterogeneous (CFTR+/-) mice were loaded with SPQ. Intracellular SPQ quench by different intracellular halide concentrations was induced with dual ionophore technique. Fo and F represent background and dye leak subtracted values of fluorescence in absence and in presence of quenching halide, respectively. Values are means ± SD.

To calibrate SPQ fluorescence in cells, the double ionophore technique, with the use of high-K+ solutions containing nigericin (5 µM) and tributyltin (10 µM), was employed (7). In these experiments, isolated crypts were loaded with SPQ and hyposmotic NO-3 (80 mM KNO3) potassium saline and then were transferred to isosmotic NO-3 (140 mM KNO3) potassium saline containing the above ionophores. Fo/Fanion values were obtained by perfusing the crypts with varying concentrations of test halide (Cl-, Br-, or I-) plus ionophore followed by SCN- in the presence of valinomycin (5 µM) to completely quench the intracellular SPQ signal and obtain basal fluorescence values. Dye leakage was ~17% of the total cellular signal over 60 min (0.0125 ± 0.008 fluorescent units/s, n = 60). Fo and Fanion values were corrected for this signal on a per-experiment basis by interpolation from the fluorescence intensity, measured in cells bathed in Cl--free medium at the onset of the experiment and at the end of the experiment or by back extrapolation from the rate of change measured during the SCN- fluorescence minimal quench signal (i.e., Fo - Fex and Fanion - Fex, where Fex = background fluorescence at the time of measurement). Both methods gave comparable values. All experimental protocols were completed within 15-20 min. The Stern-Volmer quench constants for halide quenching of SPQ in pup and adult crypts (see RESULTS) were utilized to calculate the halide influx rates in the standard experimental procedure according to the following equation: JI-=(Fo/Kq · F2) · (Delta F/Delta t), in which Delta F/Delta t is the rate of fluorescence change measured at time 0. In addition to this method, running averages of halide influx at 1.6-s intervals during the first 40 s of the fluorescence quench were calculated.

Standard experimental procedure for measuring agonist-stimulated I- influx. SPQ-loaded crypts were perfused with HEPES-buffered NaNO3 containing extracellular saline, and, after establishment of a stable fluorescence signal, bath NO-3 was replaced with I- (140 mosM). The quenched SPQ fluorescent signal (NO-3 does not quench SPQ nor compete with I-) was recorded over predefined measurement windows along the base to midcrypt axis. Basal rates of I- influx (JI-) were then calculated with the use of the Stern-Volmer quench constants for I- obtained for either mouse pup or adult crypts (see Calculation of Stern-Volmer constants for halide quenching of SPQ and RESULTS). Cl- was then reintroduced, and I- was simultaneously removed from the bath. A single agonist was administered during this recovery phase (forskolin, FSK) or after establishment of a new stable fluorescence signal (CCh or NSP4) when bath I- was reintroduced in Cl--free saline. The resulting agonist-stimulated SPQ quench curve was recorded, and JI- agonist was calculated. Agonist-induced changes in I- influx rate were then calculated from pooled values of JI- agonist and JI- basal as Delta JI- = (JI- agonist - JI- basal). In addition to this approach, mean ± SD rates of fluorescence change at 1.6-s intervals over the first 40 s of fluorescence quench were graphically displayed in histogram format as change in agonist-induced quench rate, Delta RI- = (Delta F/Delta tagonist - Delta F/Delta tbasal). This later approach allowed us to determine latency and to record changes in dye quench unrelated to initial (time 0) changes in quench rate. All SPQ-loaded crypts were superfused with saline at the same bath perfusion rate (4 ml/min); solution exchange in the experimental chamber (200 µl) occurred within 3 s.

Statistical analysis. To determine statistical significance of differences between observations within an experiment, the paired Student's t-test was used. For statistical differences between experiments, the unpaired Student's t-test was used. Between 6 and 12 separate experimental observations pooled from different dye loadings were routinely collected for each experimental condition. The Fisher's exact test was used to estimate the probability of difference for diarrheal activity measurements within CF and non-CF groups in which the tabulated frequency of occurrence was too small for chi 2 analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Effect of orally administered rotavirus and injected viral NSP4 peptide(s) in mouse pups. Inoculation of CFTR-/- mice with infectious SA11cl3 rotavirus caused diarrhea in four of five mice within 24-48 h (Table 1). When CFTR+/+ or CFTR+/- mouse pups from the same C57B1/J6 background were infected with the SA11 virus, ~78% of the mice exhibited diarrhea (Table 1). Intraperitoneal injection of intact NSP4 protein or NSP4114-135 was similarly found to elicit diarrhea in CFTR+/+ or CFTR+/- mouse pups (66 and 70% responding, respectively). CFTR-/- mouse pups inoculated with these reagents exhibited diarrhea in 50% and 43% of these mice, respectively (Table 1). Diarrheal content was graded as equally severe as that encountered in CFTR+/+ mice (3). Although the incidence of diarrhea caused by NSP4 protein or NSP4114-135 appeared lower in the CF mouse studies, there was no significant difference between CFTR+/+, CFTR+/-, and CFTR-/- littermates (Fisher's exact test for 2 × 2 contingency tables; Ref. 32). All three reagents (SA11 virus, NSP4 protein, or NSP4114-135) were effective diarrheal agents. In contrast, when infectious SA11cl3 virus was given to either adult CFTR+/+, CFTR+/-, or CFTR-/- mice, none exhibited diarrhea (Table 1).

                              
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Table 1.   Induction of diarrhea by rotavirus infection or NSP4 in CFTR-/- mice

NSP4 peptide mobilizes [Ca2+]i levels in epithelial cells isolated from the small and large bowel of both CFTR+/+ and CFTR-/- mice. Rotaviral protein production has been correlated with changes in [Ca2+]i homeostasis (39) and with Ca2+-dependent cytotoxicity during viral infection in cultured cells (33, 34). With the use of the human colonic epithelial cell line HT-29 clone 19A, which expresses plasmalemmal proteins found in both small and large intestinal cells, we recently demonstrated that exogenously added NSP4 and NSP4114-135 peptide causes [Ca2+]i mobilization by PLC-mediated IP3 production (13). We therefore investigated whether the diarrheal effects of NSP4 and NSP4114-135 peptide in vivo were also associated with native mucosal intestinal cell [Ca2+]i mobilization. Fluorescence video microscopy was used to study the effects of exogenous NSP4 addition on [Ca2+]i in intestinal crypts isolated from either small or large bowel of 8- to 13-day-old mouse pups.

Small intestinal crypts. Crypts from the small intestine were chosen in preference to villi, which dissociated during isolation. Addition of 100 nM NSP4 protein, a dose shown to promote near-maximal [Ca2+]i rises in vitro (13), elicited rapid rises in [Ca2+]i that lasted 2-5 min in CFTR+/+, CFTR+/-, and CFTR-/- small intestinal crypts. Resting [Ca2+]i values of 126 ± 28 nM (CFTR-/-, n = 13) and 110 ± 63 nM (CFTR+/+ and CFTR+/-, n = 14) increased to peak values of 205 ± 54 nM and 260 ± 116 nM, respectively (Table 2). A representative example (Fig. 2, A-C) shows consecutive images taken before, during, and after NSP4 addition to an epithelial crypt isolated from a CFTR-/- mouse. No significant difference was seen between mouse CFTR genotypes with respect to basal, peak, or net Ca2+ values (P > 0.01, Student's t-test; Table 2). These results indicated that NSP4 was an effective Ca2+-mobilizing agonist in the small intestine and that Ca2+ homeostasis, both basal and agonist-stimulated, is unaffected by the CFTR gene knockout. Not all experimental crypts responded; 27 of 108 (25%) gave [Ca2+]i rises. Increasing the dose of NSP4 from 100 to 500 nM and above did not increase the number of NSP4-responsive crypts (3 of 18 responded = 17%; data not shown).

                              
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Table 2.   Summary of the effects of 100 nM NSP4 and 100 µM CCh on [Ca2+]i levels in either CFTR-/- or CFTR+/+,+/- small and large intestine mouse crypts



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Fig. 2.   Consecutive pseudocolor images of retroviral enterotoxin protein NSP4-induced intracellular Ca2+ concentration ([Ca2+]i) rise in an isolated CFTR-deficient (CFTR-/-) small intestinal mouse crypt, loaded with fura 2. A: resting Ca2+ level of ~120 nM before agonist stimulation. B: peak Ca2+ level approaching 220 nM immediately after addition of 100 nM NSP4. C: gradual return of crypt to resting Ca2+ levels ~1.5 min after addition of NSP4.

Colonic crypts. When isolated crypts from the distal colon of CFTR+/+, CFTR+/-, and CFTR-/- mice were superfused with 100 nM NSP4 protein, [Ca2+]i rises similar to those obtained from small intestinal crypts were recorded (P > 0.1, Student's t-test; Table 2). Peak NSP4-induced [Ca2+]i values were similar in all three genetic backgrounds, although basal values were slightly, but not significantly, higher in CFTR-/- mice when all samples were pooled (P > 0.01, Student's t-test; Table 2). In our in vitro studies, we found that the NSP4-induced [Ca2+]i mobilization was susceptible to predepletion by other agonists (remaining unresponsive for tens of minutes; Ref. 13). In contrast, NSP4-induced [Ca2+]i release in both ex vivo colonic and ileal mucosal cell preparations was not desensitized by repetitive agonist administration (n = 15). In the CFTR-/- mouse crypt preparation shown in Fig. 3, identical [Ca2+]i rises were seen for consecutive NSP4 or CCh followed by NSP4 challenge. Ca2+ homeostasis in the isolated crypt therefore appears to be more tightly regulated than in colonic cell lines.


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Fig. 3.   A representative example of a single crypt. Average [Ca2+]i values from 6 measurement areas along length of an isolated CFTR-/- colonic mouse crypt, showing effect of 100 nM NSP4 (A), 500 µM carbachol (CCh, B), and 100 nM NSP4 (C). SD values are shown as error bars.

To address why the NSP4 response rate of both small and large intestinal crypts was not >25%, we quantified the responsiveness of isolated crypts to the cholinergic agonist CCh. The addition of 100 µM CCh to the bath elicited [Ca2+]i rises in a similar percentage of isolated crypts from both small and large intestine (33% responded; Table 2). Lower, more physiological doses of CCh would likely produce identical success rates for both agonists. These findings suggested that a more general cellular phenomenon, other than a lack of NSP4 receptors, was accountable for this effect. Because both M3-muscarinic receptors and the cellular response to NSP4 have been shown to be sensitive to protease digestion (13), true in vivo NSP4 potency may have been masked.

Spatial analysis of the Ca2+-mobilizing effects of NSP4 revealed no regional differences along the longitudinal axis of either colonic or small intestinal crypts. Mean resting values for base, mid, and neck regions (separated by ~25 µm) were 112 ± 142, 100 ± 25, and 102 ± 39 nM, rising to 250 ± 99, 229 ± 97, and 215 ± 79 nM, respectively (n = 29). The lack of localization-dependent effects on [Ca2+]i indicates that, regardless of the cell maturity, the membranes of epithelial cells within the crypt remained responsive to NSP4. An example of this spatial homogeneity is shown in Fig. 4. In Fig. 4, a sustained component to NSP4-induced [Ca2+]i mobilization is observed that disappeared on removal of NSP4 from the bath. Nominally Ca2+-free conditions did not affect the Ca2+ response to NSP4 protein (n = 12, data not shown), confirming that the major source of the Ca2+ rise was from intracellular stores. However, under extracellular Ca2+-free conditions, no sustained component was observed, indicating that Ca2+ influx also occurred. The effects of NSP4 ex vivo reproduced those recorded in vitro, which have been linked to the intracellular production of IP3 (13).


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Fig. 4.   [Ca2+]i measurements taken from 3 different regions separated by ~15 µm along longitudinal axis of a CFTR-/- small intestinal crypt, showing magnitude of NSP4 [Ca2+]i rise at crypt base (A), midregion of crypt (B), and near neck of crypt (C). Agonist (100 nM NSP4) was washed out of bath ~10 min after addition.

NSP4 peptide mobilizes [Ca2+]i levels in crypt epithelial cells from both pup and adult mice. Isolated colonic crypts from either normal (CFTR+/+) C57B or BALB/c mice were loaded with fura 2 and exposed to 100 nM NSP4. In the example shown (BALB/c, Table 3), NSP4 mobilized Ca2+ in crypts from both 7- to 12-day- and >25-day-old mice. Although the mean number of BALB/c crypt responses was higher than that observed in the C57B background (data not shown), there was no age dependency to the magnitude of the NSP4-induced [Ca2+]i rise (values were not statistically significant, unpaired t-test, P > 0.05, n = 13; Table 3). When CCh (100 µM) was applied after a NSP4-induced Ca2+ mobilization, 75% of pup crypts and 80% of adult crypts responded with a second [Ca2+]i rise (n = 13). In this instance, the net agonist-induced [Ca2+]i rise was significantly smaller in adult crypts (P < 0.001, unpaired t-test; Table 3). When CCh alone was applied to crypts, a similar age-dependent trend in Ca2+ mobilization was observed, which failed to be significant because of the great variability in the magnitude of the peak [Ca2+]i values (P > 0.05; Table 3). These results indicated that events distal to NSP4-elicited [Ca2+]i mobilization must regulate the age- and Ca2+-dependent Cl- secretory activity of NSP4 observed in the ex vivo mouse intestine (3).

                              
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Table 3.   Summary of the effects of 100 nM NSP4 and 100 µM CCh on [Ca2+]i levels in pup and adult BALB/c mouse colonic crypts

Calculation of Stern-Volmer constants for Cl-, Br-, and I- in adult and pup mouse crypts. To separate carrier-mediated anion transport events from conductive anion transport in SPQ-loaded crypts, the halide I- was used as a surrogate anion for Cl-. I- is not a substrate for either the Na+-K+-2Cl- cotransporter or Cl-/HCO- exchanger (36, 63). This choice was not, however, without technical drawbacks, because the Stern-Volmer relationships for Br-, I-, and SCN- in free solution are reported to become positively nonlinear at >15 mM halide (24). Nonlinear increases in Kq above this concentration suggest that, in addition to dynamic collision interactions, static interactions occur (i.e., those leading to nonfluorescent halide-SPQ complex formation; Ref. 28). Irrespective of the exact nature of this quenching (assumed to be static buffering), the dye would become noncalibrated at high halide concentrations, compromising I- usefulness as a surrogate anion for conductive Cl- influx. The Stern-Volmer relationship for Cl- on the other hand has been demonstrated to remain linear when measured both in free solution and cells and shown to be the product of the dynamic collision-quench rate constant Kq (24). Stern-Volmer relationships for Cl-, I-, and Br- were constructed in SPQ-loaded crypts with the use of the dual ionophore technique (Ref. 7; see MATERIALS AND METHODS). We found that apparent Stern-Volmer quench constants for both Br- and I-, calculated from halide concentrations of either <15 mM or <150 mM, exhibited modest <15% and slight <5% nonlinear increases in slope, respectively. This deviation was not large enough to accurately determine an additional buffering coefficient (28). Thus Kq values for Br- and I- were estimated by linear curve fitting and found to be 38.5 M-1 and 51.4 M-1, respectively.1 The Kq value for Cl- was found to be 21.5 M-1 (measurements made in both pup and adult crypts, n = 8 per halide; Fig. 1), reflecting very well the ratio of SPQ collisional quench (1:1.7:1.9) reported for colonocytes (45). These values were slightly lower than those calculated in the rabbit crypts (45) and higher than those estimated in a variety of cancer cell lines (which range between 12 M-2 and 18 M-2 depending on cell type; Ref. 58). Because we failed to measure changes in tubular geometry (shrinkage) associated with agonist stimulation when confining our measurements to basal crypt areas, we did not resort to more theoretical measurements of the Stern-Volmer constant (22). With the use of these values, 50% of cellular SPQ quench would be achieved at 26, 19.6, and 47 mM Br-, I-, and Cl-, respectively.

Calculation of basal and agonist-stimulated halide permeability. The dynamic Kq values determined above were used to calculate influx rates for the respective anions that were dependent on plasma membrane permeability and not on alterations in SPQ quench rate. Basal influx was in the order I- > Cl- > Br- (Table 4) for CFTR+/+, CFTR+/-, and CFTR-/- pup crypts. An age-dependent but not significant (P > 0.05) trend in basal influx was seen, with adult values being somewhat higher than those in pups. The lack of CFTR was associated with a statistically significant (P < 0.001) decrease in basal halide influx rate (Jhalide-) in CFTR-/- mice (Table 4).

                              
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Table 4.   Basal halide influx in CFTR-expressing (CFTR+/+,+/-) and nonexpressing (CFTR-/-) mouse pups

With the calculated Kq value for I-, we tabulated changes in agonist-evoked I- influx rate (Delta JI- agonist) in response to FSK (10 µM), CCh (500 µM), and NSP4 peptide (500 nM) in all three CFTR genomic backgrounds (Table 5). Time-based histograms of changes in agonist-induced quench rate [Delta RI- = (Delta F/Delta tagonist - Delta F/Delta tbasal)] are also shown (Figs. 5-7).

                              
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Table 5.   Summary of the effects of forskolin, CCh, and NSP4 on I - influx rates in CFTR+/+,+/- and CFTR-/- mice

FSK-stimulated I- influx into crypt cells was age independent and required CFTR. FSK, as predicted from previous studies (10, 45), induced significant I- influx in both CFTR+/+ and CFTR+/- mouse pup and adult crypts; JI- agonist increased 3.7- to 4-fold over JI- basal and were statistically significant (P < 0.001, n = 5; Table 5). Age-dependent differences in the magnitude of Delta JI- were absent (P > 0.05, n = 5; Table 5). Time-dependent analysis revealed that the peak FSK-induced quenching rate (RI- agonist) occurred 8-10 s after bath I- exchange (Fig. 5A).


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Fig. 5.   Change in SPQ fluorescence quench rate in mouse distal colon crypts elicited by cAMP-mobilizing agonist forskolin (FSK, 10 µM). A: values obtained from adult and pup CFTR+/+ and CFTR+/- mouse crypts, showing a lack of any age dependency. B: values obtained from pooled CFTR+/+ and CFTR+/- and CFTR-/- pup crypts, demonstrating that effects of FSK on SPQ quench rate were dependent on CFTR expression. Values are means ± SD. FU, fluorescence unit.

In contrast, when crypts isolated from CFTR-/- mouse pup littermates were exposed to FSK, much lower JI- agonist and Delta JI- values were obtained. In this instance, JI- agonist failed to be significantly different from basal JI- values (P > 0.05) and both Delta JI- and Delta RI- values were significantly lower than corresponding values obtained from CFTR+/+ and CFTR+/- mouse pup crypts (Delta JI- CFTR-/- = 0.03 ± 0.02 M-3/s vs. Delta JI- CFTR+/+ and CFTR+/- = 1.4 ± 0.46 M-3/s, P < 0.001, n = 5; Table 5 and Fig. 5B). The absence of CFTR was correlated with >95% inhibition of FSK-stimulated I- influx into isolated mouse pup crypts. In all genotypes, FSK stimulated changes in influx in ~90% of crypts tested.

CCh-stimulated I- influx into crypt cells was age independent and required CFTR expression. CCh (100 µM) JI- agonist values were twofold greater than JI- basal values in wild-type and heterozygous CF genotypes and were significantly different (P < 0.001, n = 5; Table 5). The resulting Delta JI- values from both groups displayed no age dependency (Delta JI- pup = 0.62 ± 0.30 M-3/s vs. Delta JI- adult = 0.69 ± 0.24 M-3/s, n = 5, P > 0.05; Table 5). These changes approximated to 51 and 40% of the values elicited by FSK in pup and adult crypts, respectively (Table 5). CCh-elicited Delta RI- changes were greatest 8-10 s after bath I- exchange, with a latency similar to that recorded for FSK (Fig. 6A). CCh was therefore a potent age-independent stimulator of plasma membrane I- influx in CFTR-containing crypts, causing identical but smaller changes in I- influx rate compared with FSK.


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Fig. 6.   Change in SPQ fluorescence quench rate in mouse distal colon crypts elicited by Ca2+-mobilizing agonist CCh (100 µM). A: values obtained from pooled adult and pup CFTR+/+ and CFTR+/- mouse crypts, showing a lack of any age dependency. B: values obtained from pooled CFTR+/+ and CFTR+/- and CFTR-/- pup crypts, demonstrating that effects of CCh on SPQ quench rate were delayed and significantly smaller in magnitude when CFTR was absent from crypt. Values are means ± SD.

Like FSK, CCh failed to significantly increase the plasma membrane I- influx rate in CFTR-/- mouse pup crypts. JI- agonist and JI- basal values failed to be statistically different (P < 0.05, Student's t-test, n = 5; Table 5). The Delta JI- recorded in CF mouse pup crypts was significantly smaller than CFTR+/+ and CFTR+/- littermate values (CFTR-/- Delta JI- agonist = 0.11 ± 0.08 M-3/s vs. CFTR+/+ and CFTR+/- Delta JI- = 0.62 ± 0.3 M-3/s, P < 0.001, n = 5; Table 5). When Delta RI- for CCh was plotted by histogram analysis in the CFTR-/- background, quite different kinetics were observed. Both the onset and subsequent peak in Delta RI- trailed wild-type and heterozygous values by 4 and 18 s, respectively (Fig. 6B). This delay was not well defined by the single exponential fitted at time 0 during initial rate analysis (JI-). Thus time-based histograms of rate change are shown. Peak values of CCh-induced Delta RI- recorded from CFTR-/- mouse pup crypts approximated to 60% of values recorded from wild-type and heterozygous genotypes. In all genotypes, CCh stimulated changes in ~65% of crypts tested. We therefore concluded that CCh, like FSK, requires CFTR to exert its major effects on plasma membrane I- influx. Rate analysis revealed, however, that a slower component of SPQ quench rate was also present when CCh, but not FSK, was added to crypts from CFTR-/- pups.

NSP4-stimulated I- influx into crypt cells was age dependent and largely independent of CFTR expression. NSP4 protein (100 nM) was found to elicit changes in plasma membrane I- influx in crypts isolated from wild-type and heterozygous mouse CFTR genotypes. However, unlike either FSK or CCh, the effects of NSP4 were clearly age dependent (Table 5). In the pup crypt, NSP4 stimulated plasma membrane I- influx by 2.7-fold, whereas, in the adult crypt, NSP4-stimulated I- influx was <1.1-fold. Corresponding P values for JI- agonist vs. JI- basal were significantly different for pup (P < 0.001) but not adult (P > 0.05) crypts. Net changes in NSP4-elicited influx rate were as follows: Delta JI- pup = 0.67 ± 0.42 M-3/s vs. Delta JI- adult = 0.06 ± 0.02 M-3/s, and were significantly different (P < 0.001, n = 5; Table 5). When the NSP4-induced change in I- influx rate was displayed in histogram format, peak changes in influx rate had a lag of 10 s after introduction of I- into the bath, similar to that recorded for both FSK and CCh (Fig. 7A).


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Fig. 7.   Change in SPQ fluorescence quench rate in mouse distal colon crypts elicited by NSP4 (100 nM). A: values obtained form pooled adult and pup CFTR+/+ and CFTR+/- mouse crypts, demonstrating that NSP4 effects were age dependent. B: values obtained from pooled CFTR+/+ and CFTR+/- and CFTR-/- pup crypts, demonstrating that effects of NSP4 on SPQ quench rate were independent of CFTR expression. Values are means ± SD.

Significantly, when NSP4 protein (100 nM) was tested on CFTR-/- mouse pup crypts, a threefold increase in I- influx rate was recorded. JI- agonist was statistically different from JI- basal (P < 0.001) and was not statistically different from JI- agonist values obtained in wild-type and heterozygous CFTR backgrounds (P > 0.05; Table 5). The net change in NSP4-induced I- influx rate approximated to 64% of CFTR+/+ and CFTR+/- littermate values (Delta JI- CFTR-/- = 0.43 ± 0.1 compared with Delta JI- CFTR+/+ and CFTR+/- = 0.67 ± 0.42). The Delta RI- values failed to exhibit either slow onset or delayed peak (as observed for CCh) in the CFTR-/- genotype (see above) but instead peaked 10 s after I- introduction into the bath, paralleling NSP4, FSK, and CCh effects in CFTR-expressing crypts (Fig. 7B). In all genotypes, NSP4 stimulated changes in I- influx in ~70% of crypts tested, a response rate twice that of NSP4-induced Ca2+ mobilization. Thus NSP4-evoked plasma membrane I- influx was age dependent, occurring selectively in mouse pup crypts, and was largely independent of CFTR expression. In crypts from wild-type and heterozygous CFTR pups, the NSP4-induced I- influx rates were larger but not significantly different (1.56-fold, n = 5; Table 5). Thus CFTR expression may additionally be utilized by NSP4 to promote greater overall changes in plasma membrane halide permeability.

NSP4-induced I- influx into pup crypt cells was dependent on [Ca2+]i mobilization. To investigate the relationship between NSP4-induced Ca2+ mobilization and plasma membrane I- influx, JI- basal, JI- agonist, and Delta JI- were measured in CFTR+/+ and CFTR+/- mouse pup crypts loaded and bathed in Ca2+-free HEPES-buffered saline (nominally Ca2+-free plus 1 mM EGTA). Basal I- influx rate was not appreciably lower in this instance than in crypts bathed with standard (1 mM CaCl2) extracellular saline (JI- basal Ca2+-free = 0.32 ± 0.08 M-3/s vs. JI- basal Ca2+-containing = 0.4 ± 0.14 M-3/s, values were not significantly different, P > 0.05, n = 4 and 15, respectively; Table 5). However, in all crypts the absence of bath Ca2+ completely inhibited NSP4-mediated I- influx (Delta JI- Ca2+-free = -0.02 ± 0.06 M-3/s vs. Delta JI- Ca2+-containing = 0.67 ± 0.42 M-3/s, P < 0.001, n = 4 and 5, respectively; Table 5). These findings demonstrated that NSP4-stimulated plasma membrane I- influx was dependent on [Ca2+]i mobilization.

NSP4 protein-induced I- influx in mouse pup crypt cells was inhibited by DIDS. The effectiveness of NSP4, but not CCh, at eliciting significant I- influx into SPQ-loaded CFTR-/- mouse pups raised the question as to the nature of this novel conductive pathway. To begin to characterize this pathway, crypts were preexposed to 0.5 mM DIDS and then washed before experimentation. DIDS inhibits a variety of Cl- channels identified in epithelial cells, including the outward-rectifying Cl- channel, the swelling-activated Cl- conductance channel, and the Ca2+-sensitive Cl- channel, but does not affect cAMP-stimulated CFTR Cl- channels (reviewed in Ref. 19). DIDS also inhibits a variety of organic osmoltye exchange mechanisms (52) and the Cl-/HCO-3 exchanger (63). Basal I- influx rate in the presence of DIDS was lower than controls but was not significantly different (JI- basal DIDS = 0.29 ± 0.13 M-3/s vs. JI- basal control = 0.4 ± 0.14 M-3/s, P > 0.05, n = 5; Table 5). This general halide transport inhibitor completely abolished the NSP4-elicited increase in plasma membrane I- influx in all crypts (JI- agonist DIDS = 0.34 ± 0.14 M-3/s vs. JI- agonist control = 01.07 ± 0.32 M-3/s, P < 0.001, n = 4), and the net change in I- influx rate reflected this difference (Delta JI- DIDS = 0.05 ± 0.06 vs. Delta JI- control = 0.67 ± 0.24, P < 0.001, n = 4; Table 5). Thus the ontogenically regulated NSP4-activated plasma membrane I- influx pathway present in mouse pup crypts was DIDS sensitive.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Relationship between the diarrhea effects of infectious rotavirus and NSP4. The induction of diarrhea by NSP4 protein and NSP4114-135 peptide compared with active rotavirus indicates that the enterotoxic effects of NSP4 may account for the initial diarrheal phase of rotaviral infection (3). During this period, diarrhea occurs without significant ultrastructural damage to the small intestinal mucosa (38, 57). This contrasts with diarrhea seen during the later stages of rotaviral infection in some animal species, in which villus blunting has been suggested to contribute to diarrhea by reducing small intestinal absorptive area (5). This later histopathological feature of rotaviral infection is not seen in all animal models; in the mouse, significant villus blunting is rare (38, 49). Therefore, loss of absorptive area is likely to be auxiliary to the underlying pathophysiological basis of diarrhea in this model. Supporting this hypothesis, we have shown that NSP4 and its active NSP4114-135 peptide fail to cause significant histological damage to the mucosa when injected intraluminally or interperitoneally into wild-type mice (unpublished observations and Ref. 3).

Correlation between [Ca2+]i mobilization and fluid transport in the gut. Addition of Ca2+-mobilizing agonists such as CCh to intestinal mucosal sheets from either CFTR-/- mice or CF patients fails to evoke a Cl- secretory current (6, 37, 53). This lack of response suggests that the cellular effects of Ca2+ mobilization on the Cl- secretory current in the gastrointestinal tract occur secondary to cAMP-dependent activation of CFTR channels and are limited to the upregulation of basolateral membrane ion transporters and K+ channels (51, 60). Results from NSP4-induced [Ca2+]i mobilization in both native small and large intestinal crypts (Fig. 2, A-C, Fig. 3, and Table 2) support the hypothesis that the enterotoxic activity of NSP4 in normal mucosa may be partly due to the secondary effects of increased cellular Cl- uptake at the basolateral plasma membrane and cellular hyperpolarization. These facilitating but not controlling mechanisms for intracellular Cl- conduction across the luminal membrane do not explain all of our observations. Two important questions relating to the role of NSP4-induced [Ca2+]i mobilization, namely the age-dependent effects of NSP4 on Cl- secretion and diarrhea and the diarrheal effects of NSP4 in CF mice, remained unanswered.

NSP4-Ca2+ signaling does not directly regulate age-dependent Cl- secretion. If changes in [Ca2+]i levels alone were solely responsible for Cl- secretion and diarrhea in mouse pups, then Ca2+ mobilization by NSP4 would be expected to be cell differentiation and/or crypt age dependent. Our studies found, however, that NSP4 mobilized [Ca2+]i equally well in all crypt regions and was therefore independent of cellular maturity (Fig. 4). These findings contrast with the reported effects of both purinergic and muscarinic agonists on [Ca2+]i in intestinal crypts, which show greater responsiveness in the basal region, where Cl- secretion is believed to occur (29, 47). The more widespread [Ca2+]i mobilization elicited by NSP4 would argue that antiabsorptive as well as prosecretory effects may be present in rotaviral-infected intestinal mucosa (see An antiabsorptive model for NSP4-induced [Ca2+]i mobilization). Furthermore, the effects of NSP4 on [Ca2+]i mobilization were neither strain dependent nor related to the age of the animals from which the crypts were isolated (Table 3). These findings therefore indicated that NSP4-mediated Ca2+ mobilization cannot directly account for the age dependency to Cl- secretion and therefore secretory diarrhea reported by us in neonatal mice (3).

NSP4-Ca2+ signaling activates an age-dependent anion permeability in normal and CF mice. Both CCh and NSP4 have been shown to utilize PLC-dependent IP3 signaling to elevate [Ca2+]i (13). We therefore examined how NSP4, but not CCh, induces diarrhea in CF mouse pups. The SPQ fluorescence imaging technique has been used previously to measure basal and agonist-stimulated Cl- permeability changes in both freshly isolated crypts (22) and primary cultures of rabbit (10, 45, 46) and human (45) distal colon. Cl- transport measurements made with SPQ have demonstrated the absence of CFTR from epithelial cells of CF patients (41, 42, 50), and modeling studies have shown that the SPQ technique provides equivalent information to that obtained from either electrophysiological or isotopic flux studies (59).

Whereas both FSK and CCh elicited age-independent increases in I- influx that are dependent on the presence of CFTR (Table 5, Fig. 5, and Fig. 6, A and B), the effects of NSP4 were age dependent and largely CFTR independent (Table 5). Both CCh and NSP4 protein were more effective at causing plasma membrane permeability changes than causing generalized [Ca2+]i mobilization. We believe this reflects both our technical limitations at isolating crypts with endogenous levels of receptor activity (see RESULTS) and the fact that Ca2+-mobilizing agonists can couple efficiently with plasma membrane anion channels through localized [Ca2+]i rises at the level of the plasma membrane that often do not provoke a generalized [Ca2+]i mobilization (35).

The lack of an age dependency for CFTR-dependent I- permeability changes corroborated Cl- transport studies made in weaning and adult rat and pig colon but differed from the age-dependent effects of cGMP or Escherichia coli heat-stable enterotoxin (STa) in primary cultures of weaning and adult rabbit colon. In these instances agonist responses were greater in crypts isolated from older mice (10). Differences may, however, reflect the fact that Cl- was used as a quencher in the latter studies, and rates may represent both conductive and nonconductive components of plasma membrane Cl- permeability. In addition, STa effects on transmucosal Cl- secretion and crypt Cl- permeability are absent in the CF mice (20). Thus NSP4 utilizes a different plasma membrane permeability pathway in distal colon crypts from that utilized by STa or its mammalian counterparts, guanylin or uroguanylin, and other nitroso compounds of nitric oxide-signaling pathways.

The age dependency to NSP4-elicited changes in plasma membrane I- influx suggests that NSP4-elicited diarrhea in CF mouse pups (Table 1) may be brought about by the activation of a novel cellular permeability pathway. The fact that neither FSK nor CCh promoted similar changes in CF mice, and that NSP4-induced I- permeation was both Ca2+ dependent and blocked by DIDS (Table 5), supported the hypothesis that we are reporting on a different permeability pathway. NSP4-induced I- influx into CFTR-/- mouse pup crypts was ~60% of that elicited in the wild-type and heterozygous background. This difference may possibly explain the higher, but not significantly different, incidence of diarrhea reported for infectious rotavirus, NSP4 protein, and NSP4114-135 peptide in CFTR+/+ and CFTR+/- mice. NSP4-induced [Ca2+]i mobilization would be expected to facilitate both CFTR-dependent and CFTR-independent anion permeation by upregulating Ca2+-dependent secondary active transport mechanisms at the basolateral membrane.

Our findings would also provide a functional explanation for the recent observations of Angel and colleagues (2), who demonstrated that CF mice and their non-CF siblings exhibit diarrhea after rotaviral infection. The age- and Ca2+-dependent changes in intestinal epithelial cell plasma membrane anion permeability evoked by NSP4, shown in this study to be CFTR independent, may contribute to (or constitute) the acute phase of the rotaviral diarrheal response. This cellular phenomena would precede immune/morphological changes occurring within the mucosa after viral infection.

Candidate anion conductances for NSP4-induced age-dependent permeability changes in the mouse pup crypt. Although the plasma membrane halide permeability changes promoted by NSP4 were clearly Ca2+ dependent and could be inhibited pharmacologically, our investigations have yet to confirm the biophysical nature of the conductance. Furthermore, the SPQ technique does not allow us to determine the plasma membrane location of the NSP4-elicited I- influx. Future studies utilizing alternate methods should address these questions. Even so, at this point, it is of interest to consider candidate conductances that may participate in this phenomena.

Direct activation of apical plasma membrane Ca2+-sensitive Cl- channels by classical PLC-dependent Ca2+-mobilizing secretagogues, although clearly demonstrated in gastrointestinal cell lines and epithelial cells from other organs, e.g., the lungs (19), has not been shown in native intestinal epithelia. However, both enteroendocrine and macrophage/lymphocyte populations of the gut are known to secrete cryptdin proteins, some of which are believed to act themselves as anion conductances (30), whereas others, notably the defensins, are proposed to act at nanomolar concentrations to stimulate Ca2+-dependent Cl- secretion (4, 31). We therefore propose that NSP4 either 1) itself creates a conductance in the apical plasma membrane, 2) activates a dormant Ca2+-activated anion channel within this membrane with biophysical properties similar to those recorded in vitro (reviewed in Ref. 19) through its stimulatory effects on PLC-induced [Ca2+]i mobilization (13), or 3) acts as a regulatory protein at the level of Ca2+- or anion-channel signaling to elicit Ca2+-dependent permeability changes across the cellular plasma membrane. CCh failed to promote similar changes in the initial rate of I- influx into CF mouse pup crypts as NSP4 but elicited a response with a delayed onset, consisting of ~20% of wild-type and heterozygous CFTR values (Table 5 and Fig. 6B). Because supramaximal concentrations of CCh were used, these differences do not reflect dose dependency and argue against the presence of an ontogenically regulated conductance that can be stimulated by all Ca2+-mobilizing agonists. The delayed onset changes in I- permeability promoted by CCh (Fig. 6B) are unlikely to be localized to the apical membrane because it is clear that CCh fails to elicit a Cl- secretory response in CF mucosal sheets (6, 37, 53).

A possibility exists that NSP4 may activate a native cell counterpart of osmotically regulated Cl- channels. These conductances, although not directly activated by Ca2+, are indirectly Ca2+ sensitive and have been shown to participate in regulatory volume decreases (RVD) in intestinal epithelia (12). These channels, believed to be localized on the basolateral plasma, participate in the maintenance of fluid and osmolyte absorption. They are functionally expressed in the surface and neck regions of the crypt as well as the small intestinal villi, areas known to exhibit RVD (11) and have been shown to be impaired in CFTR-/- mice (55). Our SPQ fluorescent measurements were made, however, in lower crypt regions, which do not undergo RVD. Thus they are poor candidates for the NSP4 response.

Volume changes in response to Ca2+-mediated secretagogues have been reported in CF mouse small intestinal crypts (56), suggesting that additional conductive pathways exist. The slow onset response observed for I- influx after CCh challenge in CFTR-/- mouse crypts may reflect this alternate pathway (Fig. 6B). However, the timing of the effects of NSP4 in the CFTR-/- mouse crypt was very different (Fig. 7B), mimicking the profile observed for activation of apical membrane CFTR in wild-type crypts by all agonists (Figs. 5A, 6A, and 7A). We interpret these results to suggest that the effects of NSP4 on any basolateral membrane permeability, if present, are likely to be secondary phenomena and unrelated to the primary effects of NSP4 on the apical plasma membrane I- influx.

A prosecretory model for NSP4-induced [Ca2+]i mobilization. A characteristic property of rotavirus infection is that it causes age-dependent gastroenteritis in neonatal mammals (25). In the neonatal mouse, increases in small and large intestinal luminal Na+, Cl-, and water content have been correlated with diarrheal activity during rotaviral infection (49). Previously, we have reported (3) that NSP4 protein addition to mouse pup small intestinal mucosal sheets mimics the transepithelial Cl- secretory effects of CCh, an endogenous Ca2+-mobilizing agonist, but that this secretory response was lost in adult tissues. Further strengthening our hypothesis that enterocytes express an ontogenically regulated, Ca2+-activated ionic conductance that underlies the diarrheal response, we now report that NSP4 induces Ca2+-dependent, DIDS-inhibitable I- influx in CFTR-/- mouse pup crypts. This phenomenon did not occur in adult crypts. The primary effect of NSP4 may therefore be to activate an apical plasma membrane Ca2+-dependent Cl- conductance in mouse pup enterocytes with biophysical properties similar to those described in vitro (19). The secondary effect of NSP4, like that of CCh, may be to facilitate CFTR-mediated fluid transport by upregulating Ca2+-sensitive basolateral Cl- uptake and cellular hyperpolarization. Combined, both may account for the Ca2+-dependent fluid secretory response of normal and CF mucosa and may delineate a secretory component to rotaviral diarrhea.

An antiabsorptive model for NSP4-induced [Ca2+]i mobilization. NSP4 mobilized Ca2+ in all regions of the crypt from both the small intestine and colon. We have also found that NSP4 is equally effective at mobilizing Ca2+ in dispersed villus epithelial cells from the small intestine (data not shown). Basal electroneutral NaCl absorption into the villus has been shown to be inhibited by Ca2+-dependent processes, including PLC-gamma , calmodulin, and protein kinase C activation (14, 17, 26). Thus another predicted secondary effect of NSP4-induced Ca2+ mobilization is downregulation of this absorptive process. Released NSP4 protein, if it encounters luminal receptors on neighboring cells (3), would be expected to promote similar antiabsorptive changes. NSP4-induced Ca2+ mobilization would not, however, be expected to affect absorptive Na+-coupled glucose-solute transport (62), which is the cellular basis of oral rehydration therapy given during bacterial and viral gastroenteritis (40).

We have shown that the nonstructural protein of rotavirus NSP4 and its peptide both elevate intestinal cell [Ca2+]i levels and induce diarrhea in neonatal mice with either wild-type or CFTR-deficient genotypes. Ca2+ mobilization may effect a diarrheal response in these cells through a variety of cellular mechanisms, including the activation of an ontogenically regulated, Ca2+-sensitive, Cl--conductive pathway in small intestinal crypts and Ca2+-inhibitable Na+-absorptive processes in small intestinal villi and crypts. Fluid loss by any of these mechanisms would occur independently of CFTR function. The diarrheal properties of NSP4 indicate that molecules based on the active domain of NSP4 may prove useful for the treatment of neonatal meconium ileus syndrome or the less severe clinical condition of constipation, which is prevalent in CF patients (44). The low dose (nanomolar) effectiveness of the NSP4 peptide and protein (3, 13), coupled with the fact that attenuated NSP4 proteins with differing diarrheal activities have been recently identified by us (64), suggests that further molecular characterization of the active domain of NSP4 may help design chemical structures with therapeutic value.


    ACKNOWLEDGEMENTS

We thank Sharon Krater for help in breeding and genotyping the CF mice. We also thank A. Beaudet, F. Murad, R. O'Neal, and J. Sellin for critical evaluation of this manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30144 (M. K. Estes), Texas Advanced Technology Program Grant 004949-062 (M. K. Estes and A. P. Morris), and Cystic Fibrosis Foundation Postdoctoral Fellowship F806 (W. K. O'Neal).

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.

1 When utilizing I- and NO-3-containing saline, it could be argued that highly membrane-permeable oxidation species (e.g., IO-3, IO-5) may explain this additional buffering component. There is no clear way of establishing an equilibrium concentration of these anions in our solutions. However, this does not invalidate this well-established fluorescent procedure because matched controls are always performed (i.e., ± agonist) and increases in Kq would serve to underestimate influx.

Address for reprint requests and other correspondence: A. P. Morris, Depts. of Integrative Biology and Internal Medicine-Gastroenterology, Medical School, Univ. of Texas at Houston, Houston, TX 77030 (E-mail: amorris{at}girch1.med.uth.tmc.edu).

Received 1 February 1999; accepted in final form 12 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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