Energy-rich phosphates and transintestinal transport in rat intestine incubated in vitro at different temperatures.

In the present work, the transported fluid and the tissue content of ATP, ADP and AMP has been evaluated in the jejunum rat intestine which was everted and incubated in vitro both at 28 degrees C and at 38 degrees C for 1 h. The energy-rich phosphates have been measured in the tissue at the beginning and at the end of the experiment as well as in vivo. These determinations have been made in the total intestine and in the scraped mucosa. ATP and ADP content are higher in vivo and lower but constant at 28 degrees C in vitro; on the contrary, at 38 degrees C in vitro, the initial and final content of these adenilic nucleotides are both lower than at 28 degrees C. Under all these conditions the AMP content does not vary appreciably. Wet weight to dry weight ratios ahve been reported for mucosal and submucosal tissues in unincubated and incubated intestines. In some experiments, fluid transport (measured as an actual serosal volume increase) was determined every 20 min during a 1-h incubation. At 28 degrees C, fluid transport is constant throughout the time of the experiment, but at 38 degrees C, there is a progressive decrease of the transported fluid. Fluid transport and ATP content of the intestine seem to be directly related. The transport activity which is lower at 38 degrees C than at 28 degrees C, seems to be due to a low availability of energy-rich phosphates.

Effect of ethyl acetate on the transport of sodium and glucose in the hamster small intestine in vitro.

The effect of ethyl acetate on Na+, water and glucose transport, as well as on glucose and electrolyte intracellular concentrations in everted and cannulated sacs of hamster jejunum, have been studied. Ethyl acetate, a substance that easily penetrates and delivers energy to the cell, strongly stimulates net glucose and Na+ transport. The explanation of the experimental results takes into account the possibility of the existence of an active extrusion of glucose at the level of the basolateral membrane of the enterocyte.

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.

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.

Characterization of basolateral membrane Na/H antiport in rat jejunum.

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte.

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.

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.

In vivo and in vitro sugar transport in frog intestine.

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.

The calculated glucose concentration profile in the intercellular spaces of everted jejunum of rat.

On the basis of experimental data from isolated and everted rat jejunum and on extension of Diamond and Bossert's mathematical model, the glucose concentration in the intercellular spaces has been calculated and has been found higher (less than 2 mM) than that present in the serosal space.

Thiamine uptake in human intestinal biopsy specimens, including observations from a patient with acute thiamine deficiency.

Mucosal biopsy specimens obtained by routine endoscopy from 108 human subjects, including one patient with thiamine deficiency, were incubated at 37 degrees C in oxygenated calcium-free Krebs-Ringer solution (pH 7.5) containing tritiated thiamine and [14C]dextran as a marker of adherent mucosal water. The amount of labeled thiamine taken up was measured radiometrically. In subjects with no clinical evidence of thiamine deficiency, 1) thiamine uptake by duodenal mucosa had a hyperbolic time course, reaching equilibrium at 10 min; 2) thiamine concentrations < 2.5 mumol/L were taken up predominantly by a saturable mechanism displaying Michaelis-Menten kinetics (K(m) 4.4 mumol/L and Jmax 2.3 pmol.mg wet tissue-1.6 min-1), whereas higher concentrations were taken up by passive diffusion; 3) thiamine transport had different capacities along the gastrointestinal tract (duodenum >> colon > stomach); and 4) thiamine uptake was competitively inhibited in the duodenum by thiamine analogs, albeit with a different order of potency compared with rats, and was blocked by 2,4-dinitrophenol. In the thiamine-deficient patient, the duodenal saturable uptake was increased, with higher K(m) and Jmax values. In conclusion, physiologic concentrations of thiamine were transported in human small intestine by a specific mechanism dependent on cellular metabolism, whose transporters appear to be down-regulated.

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.

Aging and ATPase activities in rat jejunum.

In addition to the well-known (Na,K)-ATPase activity, an ouabain-insensitive Na-ATPase has been evidenced in the basolateral membrane of intestinal and renal cells from different mammals. Basolateral membranes of jejunal enterocytes from rats of different ages, i.e., very young, young, adult and old were separated by self-orienting, Percoll-gradient centrifugation. The total protein content and both Na- and (Na,K)-ATPase activities in initial homogenate and final pellets were analyzed. The dry weight of homogenate and pellet was also determined. The two ATPase activities and the protein content of the basolateral membrane fraction decrease with age when referred to the dry weight of the pellet. This diminution is also evident in the initial homogenate. The activation curve of Na-ATPase, hyperbolic in shape, gives Km and Vmax values unaffected by aging. The same behaviour is true for the kinetic parameters of (Na,K)-ATPase, which has a sigmoidal velocity curve. From these results, it seems that both Na- and (Na,K)-ATPase have the same characteristics in the basolateral membrane of the enterocyte throughout the life span of the animal, but they decrease quantitatively with aging.

Interferon versus steroids in patients with hepatitis C virus-associated cryoglobulinaemic glomerulonephritis.

BACKGROUND/AIMS: The association between mixed cryoglobulinaemia, cryoglobulinaemic glomerulonephritis, and chronic hepatitis C virus infection has recently been described. The renal disease had usually been treated with immunosuppressive therapy, but, given the presence of viral infection, this therapy is no longer recommended. In this study, we compare steroid vs interferon therapy in a group of patients affected by hepatitis C virus-positive cryoglobulinaemic glomerulonephritis in the stationary phase. PATIENTS/METHODS: The diagnosis of cryoglobulinaemic glomerulonephritis was made bearing in mind standard criteria. Patients were randomly assigned to 2 groups receiving oral prednisone 0.2 mg/kg/die for 6 months (6 patients, group A) or lymphoblastoid interferon 3 MU, three times a week for 6 months [7 patients, group B). Hepatitis C virus-RNA was determined by reverse transcription-polymerase chain reaction and hepatitis C virus genotype according to Okamoto. Hepatitis C virus-RNA quantitation was performed by competitive polymerase chain reaction. RESULTS; The 2 groups were comparable in terms of age and severity of kidney failure. All genotypes of hepatitis C virus were found with a prevalence of Type 1b. In group A, 4 patients showed a partial response; in group B, 1 patient achieved complete remission, 4 a partial response, 2 patients in both groups showed no response. At the end of the treatment, all patients in both groups relapsed. Only 1 patient in group B became hepatitis C virus-RNA negative, and recovered from cryoglobulinaemic glomerulonephritis. CONCLUSIONS: Interferon seems to be an effective drug in the treatment of cryoglobulinaemic glomerulonephritis, but dosage and length of treatment still need to be addressed by large multicentre studies.

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.

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.

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.

Ouabain-insensitive transintestinal transport in the rat jejunum incubated in vitro.

The jejunal tract of rat intestine, everted and incubated in vitro at 28 degrees C for 2 hr in Krebs-Ringer bicarbonate solution, was used to test the existence of a ouabain-insensitive sodium pump. Cell water, Na, and K together with Na, fluid, K, and lactate transported into the serosal compartment were determined and, under control conditions, the tested parameters were found constant in time. By blocking the Na-K pump with 20 mM ouabain in the serosal compartment, the enterocyte lost K and gained Na, but the cell volume did not vary. Moreover, the transport of Na, fluid, and lactate, although lower, was constant for 2 hr. When ethacrynate was added or when the ATP supply was blocked by adding 2,4-dinitrophenol plus iodoacetate, the cell swelled and the transport of Na and fluid stopped. These results are interpreted as suggesting the existence of a ouabain-insensitive Na pump, in addition to the well-known Na-K pump.

Effects of semistarvation on transintestinal D-glucose transport and D-glucose uptake in brush border and basolateral membranes of rat enterocytes.

The present work shows that semistarvation (8-10 g of food for 10 days) increases net D-glucose, Na+ and water transport in the everted and perfused rat jejunum. A linear and positive correlation between cell sugar concentration and transport was found in control and semistarved rats, but the phenomenon was more relevant only in semistarved animals. Membrane vesicle experiments showed that semistarvation increases sugar overshoot only in brush border membrane vesicles, while this situation does not occur in basolateral membrane vesicles. The effect of partial food deprivation seems to enhance net sugar transport by increasing sugar entry across the apical membrane of enterocytes.