1 Department of Pathology, Wayne State University, Detroit, Michigan 48201; and 2 University Clinic of Surgery, Vienna General Hospital, A-1090 Vienna, Austria
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ABSTRACT |
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Na+- nutrient cotransport-dependent regulation of paracellular permeability has been demonstrated in rodent intestine and human intestinal epithelial cell lines. In cell lines this regulation is associated with phosphorylation of myosin II regulatory light chain (MLC). However, the subcellular localization of phosphorylated MLC during this regulation has not been studied and regulation of paracellular permeability and MLC phosphorylation has not been studied in isolated human intestine. To evaluate these events in human jejunum, isolated mucosa was mounted in Ussing chambers, characterized electrophysiologically, and then immunostained using anti-phosphorylated MLC and anti-total MLC antisera. MLC phosphorylation was assessed by calculating the ratio of anti-phosphorylated MLC signal to anti-total MLC signal within defined regions. Transmucosal resistance of mucosae without active Na+-glucose cotransport was 37 ± 3% greater than that of mucosae with active Na+-glucose cotransport within 15 min. Quantitative double-label immunofluorescence showed that the phosphorylated MLC-to-total MLC ratio increased by 45 ± 4% within the perijunctional actomyosin ring when Na+-glucose cotransport was active. Thus regulation of transmucosal resistance by Na+-glucose cotransport is accompanied by increased MLC phosphorylation within the perijunctional actomyosin ring. These data support the proposed critical role of the perijunctional cytoskeleton in physiological regulation of human small intestinal paracellular permeability.
myosin II regulatory light chain; tight junction; perijunctional actomyosin ring; paracellular permeability; Na+-glucose cotransporter
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INTRODUCTION |
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SMALL INTESTINAL MUCOSAL NUTRIENT absorption occurs primarily via active epithelial transport. In the case of glucose, active transport occurs at the apical, or luminal, surface of villus enterocytes via Na+-glucose cotransporter (SGLT)1 (7, 15). Glucose then traverses the basolateral membrane through the facilitated glucose exchanger GLUT2. However, it has been suggested that paracellular glucose absorption serves to augment transcellular nutrient absorption (11, 17). Consistent with this hypothesis, paracellular absorption of inert tracers is increased in the presence of active Na+-glucose cotransport (12, 18). This is also supported by mathematical modeling studies of the epithelial barrier in rat small intestine that demonstrates increased flux through small paracellular pores during active Na+-glucose cotransport (4).
Use of a reductionist Caco-2 cell model has shown that Na+-glucose cotransport-dependent regulation of paracellular permeability is associated with increased phosphorylation of the myosin II regulatory light chain (MLC), as detected in cell lysates (19). Such phosphorylation is a biochemical indicator of actomyosin contraction. On the basis of these data we hypothesized that the phosphorylated MLC might be localized to the perijunctional actomyosin ring, thereby allowing for physical interactions with the tight junction. To test this hypothesis we evaluated human jejunal mucosae that had been incubated under conditions of active or inactive Na+-glucose cotransport by double-label quantitative immunofluorescence microscopy to assess perijunctional MLC phosphorylation. The data show that decreases in transmucosal resistance, a sensitive marker of increased paracellular permeability, occur after activation of Na+-glucose cotransport and are accompanied by increased phosphorylation of perijunctional MLC in human jejunal absorptive (villus) enterocytes.
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MATERIALS AND METHODS |
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Ussing chamber experiments. Surgically excised specimens of tumor-free jejunum isolated during pancreatoduodenectomy were used. Specimens were obtained from the first jejunal segment, immediately distal to the ligament of Treitz, and the seromuscular layer was removed by blunt dissection. Mucosal preparations (1.0-cm2 surface area) from each specimen were vertically mounted in Ussing chambers (1.0-cm2 surface area; Precision Instrument Design, Lake Tahoe, CA) as described previously (13). Luminal and serosal sides were bathed at 37°C in 8 ml of nutrient buffer containing (in mM) 122 NaCl, 2 CaCl2, 1.3 MgSO4, 5 KCl, 3 glucose, 22 mannitol, and 25 NaHCO3 (pH 7.45 when gassed with 95% O2-5% CO2). Potential difference and short-circuit current (Isc) were continuously measured and recorded every 1-10 min. Luminal and serosal solutions were connected via Ag-AgCl electrodes to a voltage/current clamp (model VCC600; Physiologic Instruments, San Diego, CA). Transmucosal resistance was calculated using Ohm's law from the open-circuit potential difference and the Isc. Potential difference values were given in millivolts (lumen negative), Isc values in microamperes per square centimeter, and transmucosal resistance in ohm-square centimeters. Potential difference and transmucosal resistance values were corrected for the junctional potentials (<0.1 mV) between luminal and serosal solutions and the buffer resistance, respectively, as described previously (13). The protocol for use of human tissues was approved by the Ethics Committee of the University Clinic of Vienna (Vienna, Austria).
After 20 min of equilibration, the mucosal buffer was exchanged for buffer with 25 mM glucose (and 0 mM mannitol) or 3 mM glucose, 22 mM mannitol, and 2 mM phloridzin. Phloridzin is a glucose analog that specifically inhibits SGLT1 but is not transported into cells and does not alter intracellular glucose metabolism. Previous studies have shown that 2 mM phloridzin is sufficient to inhibit SGLT1-mediated transport of 3 mM glucose and that this does not decrease intracellular ATP levels in cultured cell lines (19). Moreover, in isolated rodent tissue, phloridzin was able to prevent glucose-stimulated SGLT1-mediated regulation of transmucosal resistance without preventing a similar fall in transmucosal resistance triggered by Na+-alanine cotransport (1), suggesting that phlorizdin is a specific inhibitor without relevant toxicities in these studies. Open-circuit potential difference and Isc were measured for 30 min, after which the jejunal tissue was snap-frozen in optimal cutting temperature solution and stored atAnti-phosphorylated MLC antibody production.
A phosphopeptide (R-P-Q-R-A-T-pS-N-V-F-A-C; pS = phosphoserine)
corresponding to serine 19-phosphorylated MLC was commercially synthesized by Mimotopes (Raleigh, NC), and 5 mg of the phosphopeptide was conjugated to 31 mg of a carrier protein, diphtheria toxoid from
the same company, as described previously (9). Two rabbits were immunized with 2.5% conjugated peptide emulsified in Freund's complete adjuvant, and immunization was boosted after 2 wk with the
same antigen solution in Freund's incomplete adjuvant. Serum from both
rabbits showed a strong reaction to MLC phosphorylated by MLC kinase
but no reactivity with nonphosphorylated MLC (Fig. 1A). The antibody was
affinity-purified with a column prepared using the SulfoLink kit
(Pierce Chemical, Milwaukee, WI) and 2 mg of the unconjugated
phosphopeptide. After elution from the affinity column fractions with
the highest protein concentrations, as determined by absorbance at 280 nm, were dialyzed against PBS with 0.05% NaN3.
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Immunostaining.
Frozen sections (3-5 µm) of the jejunal tissue were collected on
coated slides and allowed to air dry at room temperature for 1 h.
The sections were then fixed in 1% paraformaldehyde in PBS for 2 min.
After three 5-min washes in PBS, nonspecific binding was blocked by a
15-min incubation in PBS with 5% (wt/vol) bovine serum albumin.
Sections were incubated with affinity-purified anti-phosphorylated MLC
and anti-total MLC (10 µg/ml each in PBS with 5% bovine serum
albumin) overnight at room temperature in a humidified chamber. After
four 5-min washes in PBS with 5% bovine serum albumin, sections were
incubated for 1 h with Alexa 594-conjugated donkey anti-goat IgG
(Molecular Probes, Eugene, OR) diluted in PBS with 5% bovine serum
albumin. After four more washes in PBS with 5% bovine serum albumin,
unoccupied binding sites for goat IgG were blocked by incubation for 30 min in PBS with 5% normal goat serum. Sections were then incubated
with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes) and
4',6-diamidino-2-phenylindole (DAPI; 100 ng/ml) in PBS with 5% normal
goat serum for 1 h at room temperature. After four washes
in PBS with 5% normal goat serum, sections were washed once in PBS and
rinsed once in water. They were then covered with 50 µl of Slowfade
reagent in 50% glycerol (Molecular Probes), a coverslip was placed on
top, and the coverslip was sealed with nail polish. After drying the
slides were stored at 20°C. Samples stained for occludin and
phosphorylated MLC were handled as above except that monoclonal
anti-occludin (Zymed, South San Francisco, CA) and Alexa 594-conjugated
goat anti-mouse IgG were substituted for the anti-total MLC detection regents.
Fluorescence microscopy. Sections were examined under a Leica DMLB epifluorescence microscope by an observer blinded to the experimental condition using a ×63 plan apochromatic oil immersion objective, A (DAPI), I3 (Alexa 488), and N2.1 (Alexa 594) filter cubes, and a SPOT RT monochrome digital camera (Diagnostic Instruments, Sterling Heights, MI). Control sections in which one or more fluorochromes were omitted during staining were used to confirm that the signal from each fluorochrome was only detectable with the specified filter settings. Images were collected using SPOT acquisition software version 3.0.4. Exposure was controlled manually and was identical for all images of each fluorochrome. For each field photographed, separate images were collected for each wavelength.
Image analysis. Image-Pro Plus 4.1 (Media Cybernetics, Silver Spring, MD) was used to analyze the collected images. Pseudocolor images were generated by adding color to each monochrome file to create an in-register color image showing one, two, or three fluorochromes. After creation of the color image files, entire multicolor image files were deconvolved using AutoDeblur (Autoquant Imaging, Watervliet, NY). Matched regions within individual deconvolved images showing different fluorochrome combinations were cropped and enlarged using Photoshop (Adobe Systems, San Jose, CA).
For quantitative analysis of MLC phosphorylation, identical areas of interest including the perijunctional actomyosin ring of villus enterocytes were selected (by an observer blinded to the experimental condition), based on pixel locations, in nondeconvolved monochrome anti-phosphorylated MLC and anti-total MLC images. Pixel-by-pixel intensity values were arranged by x-y coordinates and exported to MS Excel (Microsoft, Redmond, WA). Data for both anti-phosphorylated MLC and anti-total MLC images were individually filtered to include only higher-intensity pixels corresponding to the perijunctional actomyosin ring, based on a minimum intensity threshold, and to allow exclusion of aberrant bright spots. Alignment of the x-y coordinates was confirmed, and the ratios of anti-phosphorylated MLC and anti-total MLC intensities were calculated for each pixel pair. These values, typically representing the ratios of 3,000-4,000 pixels, were analyzed for mean and standard deviation, and outliers were sought (none were identified). The mean of the 1,500-2,000 ratios calculated from each pair of images was then considered a single measurement. A total of 213 such pixel-by-pixel analyses were performed, 105 from jejunal mucosae incubated without phloridzin and 108 from jejunal mucosae incubated with phloridzin. Statistical evaluation of the group ratio data was performed using Excel. Group comparisons were made with Student's t-test for unpaired samples. ![]() |
RESULTS |
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SGLT1-mediated Na+-glucose
cotransport regulates transmucosal electrical resistance.
To determine whether paracellular permeability is regulated
by Na+-glucose cotransport in human small intestinal
mucosae, we measured transmucosal electrical resistance and
Isc in segments of human jejunum mounted in
Ussing chambers. Initiation of Na+-glucose cotransport led
to the generation of an SGLT1-dependent Isc of
26 ± 8 µA/cm2, consistent with transmucosal
Na+ absorption. The transmucosal electrical resistance of
mucosae with active Na+-glucose cotransport was 85 ± 5%, 73 ± 3%, and 78 ± 6% of that of mucosae with
inactive Na+-glucose cotransport after 10, 15, and 30 min,
respectively (P < 0.01 at each time point,
n = 5; Fig. 2). Thus
Na+-glucose cotransport causes changes in transmucosal
electrical resistance of human jejunum that are similar to those
previously described in isolated rodent small intestinal mucosae and
epithelial monolayers of human enterocyte cell lines (10,
19).
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SGLT1-mediated Na+-glucose cotransport is associated with increased phosphorylation of perijunctional MLC. Early studies of Na+-glucose cotransport in rodent mucosae showed a striking condensation of the perijunctional cytoskeleton in association with increases in paracellular permeability (8). We have hypothesized that this is mediated by contraction of the perijunctional actomyosin ring that, in turn, is activated by phosphorylation of MLC, a biochemical indicator of actomyosin contraction (19). However, because previous studies have only evaluated MLC phosphorylation in lysates of cultured epithelial cell lines, it has not been possible to identify the subcellular location associated with increased MLC phosphorylation after initiation of Na+-glucose cotransport. Thus, to evaluate MLC phosphorylation in intact mucosa and to determine the site of increased MLC phosphorylation, we evaluated perijunctional MLC phosphorylation in villus (absorptive) enterocytes from human jejunal mucosae that had been characterized electrophysiologically.
Our initial studies showed that the distribution of phosphorylated MLC was more restricted than that of total MLC, with the former being more greatly concentrated within the perijunctional actomyosin ring (Fig. 3). Moreover, the intensity of the phosphorylated MLC signal was increased at areas of cell-cell contact (Fig. 3). To determine whether these focal areas of increased phosphorylated MLC staining corresponded to the tight junction, we performed double-label immunofluorescence studies using the phosphospecific anti-MLC antibody and a monoclonal antibody against occludin, a transmembrane tight junction protein. Figure 4 shows that both phosphorylated MLC and occludin localize to areas of cell-cell contact. There is substantial overlap of the staining for phosphorylated MLC and occludin. Thus the cell junction-associated increases in phosphorylated MLC are partially localized within the tight junction, as defined by occludin distribution.
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DISCUSSION |
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Pappenheimer, Madara, and Reiss (8, 10, 12) demonstrated that Na+-glucose cotransport caused a coordinated regulation of tight junction structure and paracellular permeability in rodent small intestine. This increase in paracellular permeability was also associated with increases in paracellular solute flux. On the basis of these observations, the authors proposed that paracellular absorption of small nutrients contributed to overall nutrient absorption. However, despite numerous studies, this hypothesis has remained controversial. For example, endoscopic studies of human subjects failed to identify any increase in paracellular solute flux during active Na+-glucose cotransport (5, 6). Nevertheless, the in vitro observation that tight junction permeability is regulated by Na+-glucose cotransport remains, and studies using transfected epithelial cell lines have shown that tight junction regulation after Na+-glucose cotransport is associated with MLC phosphorylation (19). Although inhibitors of MLC kinase can prevent tight junction regulation in both transfected epithelial cell lines and isolated rodent mucosae (19), it has not been possible to document increases in enterocyte MLC phosphorylation in native mucosae. Moreover, because studies in cell lines have depended on cell lysates, the subcellular localization of phosphorylated MLC is unknown. Therefore, we sought to use established in vitro methods to evaluate the molecular basis of these events in intact mucosae. These studies of human jejunal mucosae show that regulation of transmucosal resistance by Na+-glucose cotransport occurs in isolated human jejunal mucosae in a manner that is both qualitatively and quantitatively similar to that in isolated rodent mucosa and cultured epithelial monolayers (1, 19). Thus we concluded that human mucosa studied in this manner represented an acceptable model of Na+-glucose cotransport-dependent regulation of transmucosal resistance.
Previous studies suggest that contraction of the perijunctional actomyosin ring, as indicated by MLC phosphorylation, is necessary for regulation of paracellular permeability in both cultured epithelial monolayers (19). However, the heterogeneity of cell types made such biochemical analyses impossible in native mucosae. We have used a phosphospecific anti-MLC antisera to evaluate MLC phosphorylation in isolated native human mucosae. This morphology-based approach allows us to limit our analysis to villus enterocytes only, without the potential for artifact introduced by other epithelial and nonepithelial cell types. Quantitative analysis demonstrated a 45 ± 4% increase in the phosphorylated MLC-to-total MLC ratio at the perijunctional actomyosin ring as a consequence of active Na+-glucose cotransport. Moreover, these studies show that phosphorylated MLC is concentrated within the perijunctional actomyosin ring and is, therefore, physically linked to the tight junction, the primary determinant of paracellular permeability. Notably, the distribution of phosphorylated MLC was more restricted than that of total MLC and was enhanced in areas that, corresponded to the tight junction.
The molecular details responsible for the regulation of tight junction permeability via actomyosin contraction remain largely unknown, but the functional and structural association of the tight junction with the perijunctional actomyosin ring is well recognized (16). Moreover, a number of interactions between tight junction proteins and actin have been described. In light of these biochemical data, ZO-1, a peripheral membrane protein localized to the cytoplasmic face of the tight junction (14), may be involved in the effects of MLC phosphorylation on tight junction permeability. ZO-1 is a member of the PDZ family of proteins that contain domains specialized to mediate for protein-protein interactions and has both actin binding and actin cross-linking properties (20). Other tight junction proteins, including ZO-2, ZO-3, and occludin, also bind to actin (20), and the tight junction protein cingulin interacts with ZO-1, ZO-2, and ZO-3, as well as enterocyte myosin heavy chain (3). On the basis of these in vitro data, numerous functional interactions between the tight junction and perijunctional actomyosin ring are possible. However, at present, analysis of these interactions within epithelial cells is lacking.
Previous analyses of rodent mucosae have shown that capacitance and conductance are both increased by Na+-glucose cotransport, indicative of increases in membrane surface area and width of intercellular junctions, respectively (10). This is consistent with contraction of the perijunctional actomyosin ring, as would be triggered by MLC phosphorylation. However, we were unable to detect decreases in the dimensions of the perijunctional actomyosin ring. We were also unable to detect any changes in inter-tight junction dimensions (as delineated by immunostaining with antibodies to the tight junction proteins occludin and ZO-1; unpublished observations). Nevertheless, the lack of gross changes in these dimensions is not surprising, because previous ultrastructural studies were also unable to detect changes in these cell dimensions (2, 8).
In summary, these studies show for the first time that the paracellular permeability of human jejunum, as demonstrated by transmucosal resistance, is regulated by Na+-glucose cotransport. This regulation is associated with increases in MLC phosphorylation within villus absorptive enterocytes. The localization of these increases in perijunctional MLC to the perijunctional actomyosin ring, with enhanced MLC phosphorylation at the apical junctional complex, supports a model of cytoskeletally mediated tight junction regulation.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Yasuharu Sasaki, Katsuhiko Sakurada, and Fumio Matsumura for sharing the anti-phosphorylated MLC antibodies that were used in our preliminary studies.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02503 and DK-56121.
Address for reprint requests and other correspondence: J. R. Turner, Dept. of Pathology, Univ. of Chicago, 5841 South Maryland Ave., Mail Code 1089, Chicago, IL 60637 (E-mail: jturner{at}bsd.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 June 2001; accepted in final form 14 August 2001.
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