Effects of creatine in a rat intestinal model of ischemia/reperfusion injury.

PURPOSE: Creatine belongs to a buffering system of cellular ATP level and has been reported to display direct antioxidant activity. Aim of this work was to investigate whether creatine treatment could ameliorate the antioxidant response of intestinal cells and limit the oxidative injury induced by anoxia and subsequent reoxygenation. METHODS: Jejunal and ileal tracts of rat intestine were everted and incubated in vitro under normoxic, anoxic and reoxygenation conditions in the absence and in the presence of 10 mM creatine. (Na+, K+)-ATPase, γ-GT and antioxidant enzymes activities were determined in mucosal homogenate, as well as malondialdehyde production and HSP70 expression. RESULTS: Both in jejunum and ileum, creatine treatment increases (Na+, K+)-ATPase activity; γ-GT is unaffected in jejunum but stimulated in ileum. In both tissues, creatine does not alter the antioxidant activities or malondialdehyde level. HSP70 expression is increased only in jejunum. Anoxic conditions stimulate antioxidant activities to a greater extent in jejunum compared to ileum; reoxygenation does not evoke further effects, but enhances malondialdehyde production in both tracts. The protective action of creatine, in reoxygenation, is more marked in jejunum as for its stimulation of antioxidant activities; however, in jejunum, a prooxidant action of creatine is suggested, since malondialdehyde production is enhanced by its presence; on the contrary in ileum, where HSP70 is overexpressed in reoxygenation, peroxidation level is significantly reduced. CONCLUSIONS: The presence of creatine seems to potentiate the defensive response of both tissues, in jejunum by means of cell antioxidant equipment, in ileum by the involvement of HSP70.

Consumidores de cocaína: del uso recreativo al consumo adictivo. Una propuesta de intervención preventiva y asistencial / Cocaine takers: from recreational use to addictive consumption. A proposal for preventive and health-care intervention

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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.

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 structural 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 muM 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.

An endogenous monocarboxylate transport in Xenopus laevis oocytes.

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes. (36)Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement. L-[(14)C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na(+) in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [(14)C]pyruvate uptake exhibited the same pattern of functional properties evidenced for L-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.

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.

Energy depletion differently affects membrane transport and intracellular metabolism of riboflavin taken up by isolated rat enterocytes.

Isolated rat enterocytes, both normal and those de-energized with rotenone, were used to study the energy dependence of membrane and intracellular intestinal riboflavin transport in vitro. Membrane and intracellular transport were investigated by using short (3 min) and long (20 min) incubation times, respectively. For both types of cells and incubation times, [3H]-riboflavin uptake presented a saturable component prevailing at physiologic intraluminal concentrations. At 3 min incubation, saturable [3H]-riboflavin transport was apparently an energy-independent process with high affinity and low capacity. Values of the saturable component and its apparent constants, Km and Jmax, did not differ in normal and de-energized enterocytes. At 20 min incubation, saturable [3H]-riboflavin transport was a strictly energy-dependent process in which values of the saturable component were significantly greater in normal than in de-energized enterocytes. Km values did not differ in the two types of cells and were unmodified over 3 min, whereas in normal enterocytes, Jmax at 20 min [6.25 +/- 0.2 pmol/(mg protein. 20 min)] was significantly greater than at 3 min [2.67 +/- 0.33 pmol/(mg protein. 3 min)] and compared with de-energized enterocytes at 20 min [2.54 +/- 0.16 pmol/(mg protein. 20 min)]. Both membrane and intracellular events were inhibited by unlabeled riboflavin and analogs, which are good substrates for flavokinase, thus demonstrating the paramount role of this enzyme in riboflavin intestinal transport.

pH dependence of Cl/HCO3 exchanger in the rat jejunal enterocyte.

During bicarbonate absorption in rat jejunum, a Cl/HCO3 exchanger mediates bicarbonate extrusion across the basolateral membrane of the enterocyte. Previous studies demonstrated that anion antiport exhibits a particular behaviour: its activity is positively affected by the presence of sodium, but the cation is not translocated by the carrier protein. In view of the particular features of the jejunal Cl/HCO3 antiporter, first we performed a pharmacological characterisation of the transport protein using various Cl channels blockers. Then, since it is well known that anion exchangers play a substantial role in cell pH regulation, we investigated the possible involvement of jejunal basolateral Cl/HCO3 antiporter in intracellular pH maintenance. The sensitivity of the exchanger to pH was investigated by measuring 36Cl uptake into basolateral membrane vesicles either varying simultaneously intra- and extravesicular pH, or presetting at 7.4 external pH and varying only the internal one. Experiments were performed both in the absence and in the presence of Na. In all the tested conditions, uptake peaked at pH of about 7. 3-7.4 and then decreased, suggesting that the main function of Cl/HCO3 exchanger is related to HCO3 absorption rather than to intracellular pH control. Since pH-regulating mechanisms counteracting acidification are well known in the jejunal enterocyte, we investigated how it regulates pH after alkalinisation of the cytosol. We tested both basolateral and brush border membrane vesicles for the presence of a K/H exchanger, but we could not give evidence for its presence by means of 86Rb uptake experiments. In conclusion, the jejunal enterocyte seems to lack a mechanism counteracting cellular alkalinisation: the main purpose of pH homeostasis might be to hinder acidification of the cytosol due to influx of protons and production of acid by the metabolism.

Cl/HCO3 antiport affects H-lactate symport activity at the basolateral pole of jejunum enterocyte.

BACKGROUND/AIMS: Under normal conditions the jejunal tract of the rat intestine absorbs HCO3. A basolateral Cl/HCO3 exchange, evidenced by means of membrane vesicles, could be involved in this process. Aim of this study was to investigate the anion exchange activity in the whole jejunal tract, where various transport systems could interact. METHODS: In the jejunal tract of rat intestine everted and incubated in vitro, the experimental conditions set up minimized loss of CO2 from the serosal solution, where pH and pCO2 were determined together with fluid and electrolyte transintestinal transport. RESULTS: The serosal pCO2 increase and pH decrease, evident during the experiment, could be antagonized by enhancing the 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) concentrations in the serosal fluid. Moreover high DIDS concentration affected fluid, sodium, lactate, bicarbonate and, although in the opposite direction, chloride transport, whilst they were ineffective on K flux. CONCLUSION: These results give evidence that in the basolateral membrane the inhibition of Cl/HCO3 antiport causes a diminution of lactic acid movement. Therefore we can hypothesize that Cl/HCO3 antiport facilitates basolateral H-lactate symport in order to carry endogenous lactic acid towards the blood stream.

Functional expression of basolateral Cl-/HCO3- exchange from rat jejunum in Xenopus laevis oocytes.

Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl-/HCO3- antiport was investigated by means of 36Cl- uptake. Two days after injection of 50 ng of poly(A)+ RNA, Cl- uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0.2 mM DIDS, whereas endogenous Cl- uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl- uptake was detected with mRNA size fraction of about 2-4 kb in length. The expressed Cl- uptake can occur against a Cl- concentration gradient and is unaffected by the known Cl- channel blocker anthracene-9-carboxylic acid. Cl- transport mechanism has properties similar to jejunal basolateral. Cl-/HCO3- exchange with regard to Na+ dependence.

Facilitated transport of lactate by rat jejunal enterocyte.

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.

Proton-lactate cotransport in basolateral membrane vesicles from rat jejunum.

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H+ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN-: this finding could explain results recently reported in the literature in which H(+)-lactate symport was not evidenced in basolateral membranes from rat jejunum.

Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum.

The jejunal basolateral Cl/HCO3 exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO3 exchanger.

Cl/HCO(-3) exchanger is operative in isolated enterocytes from rat jejunum.

Enterocytes isolated from rat jejunum were tested for the existence of a Cl-/HCO(-3) exchange, previously evidenced in basolateral membrane vesicles but not in brush border. Cells were found to retain functional integrity and transport capabilities long enough to allow Cl- fluxes to be measured. Both efflux and uptake experiments indicate that a Cl-/HCO(-3) antiport, inhibited by 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), is functional under resting conditions.

Bicarbonate and chloride transport across rat ileal basolateral membrane.

The mechanisms of HCO3- and Cl- transport across basolateral membranes from rat ileum were investigated in isolated vesicles by means of uptake experiments. Neither Cl-/HCO3- exchanger nor Na(+)-(HCO3-)n cotransport seem to be present in ileal basolateral membranes. Moreover Cl- uptake is unaffected by cis Na+ and/or K+ gradients, indicating the absence of Na(+)-Cl-, K(+)-Cl- and Na(+)-K(+)-2Cl- symport activity. An electrically conductive pathway seems to be responsible for both HCO3- and Cl- fluxes. Evidence is also given for the presence of a Na+/H+ exchanger at the basolateral pole of ileal enterocytes.

Rat jejunal basolateral membrane Cl/HCO3 exchanger is modulated by a Na-sensitive modifier site.

A Cl/HCO3 exchanger mediates HCO3 extrusion across rat jejunal basolateral membrane. Previous studies demonstrated that anion antiport activity is positively affected by Na, but evidence was given that this cation is not translocated by the carrier protein. Basolateral membranes isolated from rat jejunum were used to give more insight on Na effect. Uptake studies, performed together with vesicle sidedness determinations, indicated that the greatest stimulation of Cl-dependent HCO3 uptake occurs when Na is present at both vesicle surfaces. The kinetic dependence of Cl/HCO3 exchange on equal intra- and extravesicular Na concentration showed a hyperbolic relationship, and the calculated kinetic parameters were Vmax = 0.153 +/- 0.006 nmol mg protein-1 sec-1, Km = 23.0 mM. Ion replacement studies indicated that Na can be partially substituted only by Li and not by other monovalent cations. Results of this study suggest that Na could act as a nonessential activator of the Cl/HCO3 exchanger. A possible role of the Na-sensitive modifier site in the physiology of jejunal enterocyte is suggested.

Basolateral Cl/HCO3 exchange in rat jejunum: the effect of sodium.

Basolateral membrane vesicles isolated from rat jejunum were used to characterize a Cl/HCO3 exchange mechanism previously evidenced. Cl uptake experiments provided no evidence for Cl/OH countertransport, confirming anyhow the presence of Cl/HCO3 antiport, which was inhibited by 2 mM furosemide and unaffected by 2 mM amiloride. An outwardly directed Na gradient stimulated Cl uptake and this effect was increased if Na was present at both vesicle surfaces. To investigate the mechanism of coupling between Na and the transport protein, we performed Na uptake experiments. Na uptake was unaffected by cis-bicarbonate and trans-Cl gradients; the reversal of anion gradients was still ineffective. Similar results were obtained when a pH difference across the membrane vesicles was imposed. This study seems to suggest that Na is not transported by the Cl/HCO3 exchanger and that another mode of Na dependence must be taken into account.

Basolateral Cl-/HCO3- exchange in rat jejunum: evidence from H14CO3- 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.

D-glucose transport systems in rat jejunal brush border membrane: influence of ageing.

Jejunal brush border membranes were isolated from rats of different ages (very young, young, adult and old); the gamma-GT specific activity and the vesicle volumes were unaffected by ageing, whilst protein content was significantly reduced in brush border from old rats. Vesicles were used to investigate the kinetics of Na-glucose cotransport under voltage-clamped and zero-trans conditions over a wide range of D-glucose concentrations (0.005-70 mM). Results provide evidence that in all the ages tested D-glucose can cross the brush border membrane both by a passive diffusional component and by two Na-dependent saturable transport systems, namely one with high-affinity and low-capacity and the other with low-affinity and high-capacity. However, in some old rats only one saturable and a very small passive component occur. The two Na-dependent transport systems were analyzed to define the stoichiometry of coupling between Na and glucose fluxes. In all the ages tested the Na:glucose ratio is higher in the high-affinity system than in the low-affinity one. Accordingly the effect of a superimposed membrane potential is more evident for the high-affinity transport mechanism. In conclusion, D-glucose transport systems seem to be unaffected by ageing from very young to adult rats; only in old animals age-related alterations can be observed.