Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Morphology of the differentiation and maturation of LLC-PK1 epithelia

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC-PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na(+)-dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC-PK1 differentiation, a quantitative morphologic-stereologic-investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC-PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities (Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V) and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC-PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na(+)-dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC-PK1 monolayers.     

Development and polarization of the Na+/H+ antiport system during reorganization of LLC-PK1A cells into an epithelial membrane

Changes in Na+/H+ antiport activity and transepithelial electrical resistance were analyzed in a clone of LLC-PK1 cells as the dispersed cells became organized into an epithelial membrane. The clone designated LLC-PK1A showed a 250% increase in Na+/H+ exchange activity as compared with the parent cell line. Na+ influx induced by an outwardly oriented H+ gradient is almost completely abolished during active cell proliferation or after cell dispersion. The activity of the Na+/H+ antiport system increases after plating the cells at high density. This increase precedes the increase in the transepithelial electrical resistance. The increase in the Na+/H+ antiport activity was not observed when the cells were plated at low density in the presence of an antimitotic agent indicating that close cell contact is an absolute requirement for the development of the system. The increase in Na+ influx correlated with an increase in Vmax, while the Km for Na+ remained essentially unchanged. Unidirectional Na+ influx measured from the apical or basolateral side as the dispersed cells became reorganized into an epithelial membrane indicated that the insertion of the Na+/H+ antiporter proteins occurred directly in the apical membrane of the epithelial cells. This finding is consistent with the hypothesis that the sorting of native proteins occurs intracellularly prior to their insertion in the apical membrane of the epithelial cells. The delay in the increase of transepithelial electrical resistance as compared with the increase in Na+ influx indicates that the settlement of the limits between the apical and basolateral membrane (fence function) precedes the closing of the intercellular space (barrier function) during the development of the occluding junctions. Further, the development of the Na+/H+ antiporter was inhibited by cycloheximide but not by actinomycin D, suggesting that the expression of epithelial cell polarization is a translational or posttranslational event.     

Transport systems for polyamines in the established renal cell line LLC-PK. Polarized expression of an Na(+)-dependent transporter.

We present evidence for the existence of an Na(+)-dependent transporter and an Na(+)-independent transporter for polyamines in LLC-PK1 cells. Both transporters could be discriminated by their sensitivity to inhibitors, particularly rho-chloromercuriphenyl sulphate and various polycationic molecules. By using cell monolayers grown on a permeable filter support, we have found that the Na(+)-dependent polyamine uptake occurred preferentially from the basolateral side. The Na(+)-independent uptake, on the other hand, occurred to the same extent from either the apical or the basolateral side.     

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm

Na+,K(+)-ATPase is a marker of the basolateral plasma membrane domain of polarized epithelial cells, including the mural trophectoderm of the mammalian blastocyst (Watson and Kidder (1988). Dev. Biol. 126, 80-90). We have used this marker to explore the factors governing the establishment and maintenance of apical/basolateral polarity during differentiation of trophectoderm. A polyclonal antiserum (anti-GP80) against human cell-CAM 120/80, a homolog of the mouse cell-cell adhesion protein, uvomorulin, was used to prevent cell flattening (compaction) and formation of the epithelial junctional complex. The majority of treated embryos failed to develop a blastocoel; instead their blastomeres developed fluid-filled cavities that expanded while untreated control embryos were cavitating. Immunocytochemistry revealed that the catalytic subunit of Na+,K(+)-ATPase was contained within the membranes lining these cavities, as well as within numerous punctate foci in the cytoplasm. The down-regulation of expression of the enzyme that normally occurs in the ICM and polar trophectoderm did not take place, since the immunoreactivity remained equally strong in all blastomeres. The enzyme could not be detected in plasma membranes. We conclude that uvomorulin-mediated cell adhesion is involved in spatially restricting the expression of the catalytic subunit and is a prerequisite for the insertion of enzyme-laden vesicles into plasma membranes, but not for expression of the catalytic subunit gene. When fully developed blastocysts were treated with cytochalasins to disrupt the epithelial junctional complex, the catalytic subunit shifted from the basolateral to the apical plasma membrane. This finding suggests a primary role for the apical plasma membrane in the process of polarization, and implies that tight junctions are a manifestation of polarity that serve to maintain the separation between apical and basolateral markers.     

Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+- ATPase, a basolateral membrane protein, was not affected by drug- induced depolymerization of MTs. These observations indicate that Golgi- derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT- organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

Physiological characterization of the Na(+)/Ca(2+) exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.     

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.     

Uptake of aminoglycoside antibiotics into brush-border membrane vesicles and inhibition of (Na+ + K+)-ATPase activity of basolateral membrane

The effects of aminoglycoside antibiotics on plasma membranes were studied using rat renal basolateral and brush-border membrane vesicles. 3',4'-Dideoxykanamycin was bound to the basolateral membrane and brush-border membrane vesicles. They had a single class of binding sites with nearly the same constant, and the basolateral membrane vesicles had more binding sites than those of the brush-border membrane. Dideoxykanamycin B was transported into the intravesicular space of brush-border membrane vesicles, but not into that of basolateral membrane vesicles. The (Na+ + K+)-ATPase activity of the plasma membrane fraction prepared from the kidney of rat administered with dideoxykanamycin B intravenously decreased significantly. Aminoglycoside antibiotics entrapped in the basolateral membrane vesicles inhibited (Na+ + K+)-ATPase activity, but those added to the basolateral membrane vesicles externally failed to do so. The activity of (Na+ + K+)-ATPase was non-competitively inhibited by gentamicin. It is thus concluded that aminoglycoside antibiotics are taken up into the renal proximal tubular cells across the brush-border membrane and inhibit the (Na+ + K+)-ATPase activity of basolateral membrane. This inhibition may possibly disrupt the balance of cellular electrolytes, leading to a cellular dysfunction, and consequently to the development of aminoglycoside antibiotics' nephrotoxicity.     

K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells

Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl(-) cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase alpha1-subunit (alpha1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous alpha1NaK, and it recruited alpha1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of alpha1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with alpha1NaK. In conclusion, KCC3a forms a functional complex with alpha1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates alpha1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na(+)-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3-O-methyl-d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na(+)-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na(+)/K(+)-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na(+)-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na(+)/K(+)-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na(+)-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.