A. Faelli

Expression and immunolocalization of aquaporin-7 in rat gastrointestinal tract.

Background information. In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase—PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry.

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.

Intestinal sugar transport during ageing

Ageing effects on sugar intestinal transport were studied by using the everted sac and the brush-border membrane vesicle techniques. Four age groups of rats were used: very young, young, adult and old animals. Net transintestinal transport of D-glucose and intracellular sugar accumulation were greater in young than in very young, adult and old rats. Net Na+ transport was high in very young and young animals and then it declined with age. In brush-border membrane vesicle experiments D-glucose overshoot was smaller in the groups of animals where net sugar transport was less. In old rats, however, the overshoot did not occur. Short-circuiting of vesicles with valinomycin showed that the driving forces for sugar accumulation, i.e. the chemical potential gradient of Na+ and the electrical potential gradient, played different roles during ageing. In very young animals the chemical potential gradient seems to be responsible for D-glucose overshoot; in young rats both gradients are important while in adult animals the electrical potential gradient represents the main driving force.

Sugar and electrolyte absorption in the rat intestine perfused “in vivo”

Summary1.Studies are reported of rat intestinal perfusion “in vivo” in which data relating to the transport of sugars, sodium, fluid as well as intracellular concentrations of sugars and electrolytes have been obtained.2.A direct linear relationship with a zero intercept between net sodium and net sugar transport has been observed.3.The average intracellular concentration of sugars is always lower than in the luminal fluid and blood; this fact is consistent with the existence of a sugar pump located at the serosal pole of the absorbing cell.4.Intracellular sodium concentration is lower and intracellular potassium concentration is higher than in “in vitro” experíments.5.Cell sodium concentration and content as well as cell water content are increased by raising the luminal glucose concentration. In the same situation cell potassium concentration decreases while cell potassium content remains unchanged.6.Transport phenomena are more efficient in “in vivo” than in “in vitro” experiments.

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.

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.