M. Tosco

The basolateral membrane of rat enterocyte: Its purification from brush border contamination

Basolateral membranes obtained by self-orienting Percoll-gradient centrifugation were treated with 5 mM CaCl2 to minimize the cross-contamination by brush border membranes. From marker enzyme-specific activities it was calculated that in this preparation the basolateral/brush border membrane ratio was 22.6. A low L-glucose permeability across basolateral membrane vesicles together with ATP-dependent sodium uptake was observed.

Functional Characterization of Wild-Type and a Mutated Form of SLC26A4 Identified in a Patient with Pendred Syndrome

Background: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. Methods: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4S28R) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. Results: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. Conclusions: The functional characteristics of SLC26A4S28R we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived.

Cl/HCO3 exchange in the basolateral membrane domain of rat jejunal enterocyte

SummaryBasolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO3 movements. Uptake experiments provided no evidence for a coupling between Na and HCO3 fluxes; K−HCO3 and K−Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO3 exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO3-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO3 antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.

A MONOCARBOXYLATE TRANSPORTER MCT1 IS LOCATED AT THE BASOLATERAL POLE OF RAT JEJUNUM

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton‐lactate symporter was investigated by means of L‐[14C]lactate uptake. The existence of an endogenous capacity for L‐lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L‐lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH‐dependent lactate uptake was detected with the mRNA size fraction of about 2‐3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase‐polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

BackgroundSeveral aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.ResultsRT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding.Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein.The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.ConclusionThe results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

A creatine transporter is operative at the brush border level of the rat jejunal enterocyte

Although ergogenic effects and health benefits have been reported for creatine used as nutritional supplement, to date little is known about the mechanism of creatine absorption in the small intestine. Thus the current study was undertaken to elucidate the mechanism of creatine intake in rat jejunum with the use of well-purified brush border membrane vesicles, isolated from jejunal enterocyte. Creatine uptake was found markedly stimulated by inwardly directed Na+ and Cl− gradients, potential-sensitive, strongly reduced by the substitution of Na+ and Cl− with various cations and anions and positively affected by intravesicular K+. Moreover, creatine uptake is: 1) significantly inhibited by creatine stuctural analogs, 2) abolished by low concentrations of 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), 3) saturable as a function of creatine concentration with an apparent Michaelis-Menten constant of 24.08 ± 0.80 μM and a maximal velocity of 391.30 ± 6.19 pmoles mg protein−1 30 s−1. The transport is electrogenic since at least two Na+ and one Cl− are required to transport one creatine molecule. Western blot analysis showed the same amount of creatine transport protein in the jejunal apical membrane when compared to ileum. Thus, these data demonstrate the existence of a Na+- and Cl−-dependent, membrane potential-sensitive, electrogenic carrier-mediated mechanism for creatine absorption in rat jejunal apical membrane vesicles, which is biochemically and pharmacologically similar to those observed in other tissues. However, in other cell types the stimulatory effect of intravesicular K+ was never detected.

Basolateral Cl−/HCO−3 exchange in rat jejunum: Evidence from H14CO−3 uptake in membrane vesicles

Bicarbonate transport across basolateral membrane vesicles from rat jejunal enterocyte was studied at 28 degrees C and pH 8.2. These experimental conditions make possible the determination of [14C]bicarbonate uptake. Inward gradients of Na+, K+, and Li+ did not stimulate HCO3- uptake, suggesting that a cotransport mechanism with these cations does not occur. On the contrary a countertransport of bicarbonate driven by a Cl- gradient was evidenced. The ability of other inorganic anions to exchange with HCO3- was examined and results indicate that Cl- can be substituted by NO3-, Br- and SCN-. The Cl(-)-dependent HCO3- uptake was strongly inhibited by SITS and DIDS, whereas acetazolamide was ineffective: thus transfer of labelled CO2 is eliminated as a possible mode of HCO3- permeation. HCO3- uptake was also affected by the presence of superimposed membrane potentials, suggesting that a HCO3- conductive pathway is present in the jejunal basolateral membrane. These results show that there are no fundamental differences between data obtained performing H14CO3- and 36Cl- (previously reported) uptake experiments.

Jejunal creatine absorption: what is the role of the basolateral membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Extracellular space determination in rat small intestine by using markers of different molecular weights.

The apparent extracellular space (ECS) of rat jejunum, everted and cannulated “in vitro”, has been measured by using extracellular markers of different molecular weights. The markers used were two polyethyleneglycols,14C and3H labelled (14C-PEG MW 4000 and3H-PEG MW 900) and3H-sucrose. The ECSs for the mucosal and serosal sides have been separately determined throughout the time course, and it has been found that the two spaces are identical when PEG 4000 was used but the serosal ECS is almost the double when using PEG 900. The serosal ECS determined with sucrose is four times as big as the mucosal ECS. It seems reasonable to conclude that the best marker for the measure of total apparent ECS is sucrose, placed in the serosal compartment, taking into account that the mucosal ECS is four times smaller than the serosal one. All the markers used reach equilibrium with ECS, more rapidly in the mucosal than in the serosal ECS.Finally, by comparing cell water and cell Na concentrations, one observes that there is a statistical difference between the results obtained by using PEG 4000 as an extracellular marker and those obtained with sucrose.

Sodium transport in basolateral membrane vesicles from rat enterocytes

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.