The effect of BaCl2 on intestinal sugar transport in the rat in vitro

The effect of BaCl2 on galactose transport across isolated rat small intestine has been investigated. The addition of 5 mM BaCl2 or theophylline (3 mM) to the bathing solutions increased cell water free sugar accumulation and decreased mucosal to serosal sugar fluxes. However the effects of BaCl2 were smaller than those induced by theophylline. Removal of Ca2+ from the bathing solutions did not modify the response to BaCl2, though the response to theophylline was partially reduced. In the presence of 0.1 mM trifluoperazine, both theophylline and BaCl2 were without effect on sugar transport. These findings are discussed in terms of an effect of Ba2+ on intestinal smooth muscle tone.     

Short-chain fatty acids enhance diffusional ca transport in the epithelium of the rat cecum and colon

We examined the effect of short-chain fatty acids (SCFAs) on Ca absorption from the large intestine in rats in vitro. An Ussing-type chamber technique was used to determine the net transport of Ca from the luminal side to the basolateral side of isolated epithelium in cecum and colon preparations. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. Both solutions were warmed at 37 degrees C and bubbled with 95% O2 and 5% CO2. During and after the incubation period (30 min or 60 min), the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was evaluated. The addition of 80 mM acetic acid, 40 mM propionic acid and 10 mM butyric acid to the mucosal medium increased net Ca absorption (about 300%) in the cecum and colon. An individual application of acetic, propionic or butyric acid (0.01 to 100 mM) to the mucosal medium also increased net Ca absorption at doses of 10 mM and /or 100 mM in the cecum and colon. An increase in solute concentration in the mucosal medium by addition of glycerol or PGE400, or a decrease in pH (7.0-3.0) by addition of HCl did not affect transepithelial Ca transport. We concluded that SCFAs affect the epithelial tissue and promote Ca absorption from the large intestine in vitro. The enhancement of Ca transport induced by SCFAs might be involved in the paracellular transport mechanism.     

Role of calcium in the phloretin effects on sugar transport in rat small intestine

The effect of phloretin on D-galactose transport in rat small intestine has been investigated. Phloretin enhanced tissue sugar accumulation and reduced mucosal to serosal D-galactose fluxes. Calcium-deprived bathing solutions and verapamil significantly reduced, but did not abolish, the phloretin-effects on intestinal galactose transport. Furthermore in the presence of the anticalmodulin drugs, RMI 12330A and trifluoperazine, phloretin was without effect on D-galactose transport. These findings suggest that phloretin may reduce serosal sugar permeability via an increase in Ca2+-calmodulin complex.     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

Intestinal glucose and galactose transport in the cultured gilthead bream (Sparus aurata)

1. Electrical parameters and transepithelial glucose and galactose transport were determined in vitro across anterior and posterior intestine of the culture fish Sparus aurata. 2. Electrical potential difference (PD) and short-circuit current (Isc) were serosa-positive in anterior intestine, while they were serosa-negative or near zero in posterior intestine. 3. Tissue conductance (Gt) was higher in posterior than in anterior intestine. In both parts it was decreased when the Na ion was omitted in mucosal and serosal reservoirs. 4. Addition of glucose or galactose to the mucosal side of intestine caused an increase in PD and Isc in posterior intestine but did not significantly change PD and Isc in anterior intestine. 5. Isotopic flux of glucose and galactose measurements in short-circuit conditions showed a net active glucose and galactose absorption in posterior intestine, while in anterior intestine active transport of glucose or galactose was not observed. 6. The net transport of glucose and galactose in posterior intestine was decreased to zero in the absence of Na in mucosal and serosal reservoirs or in the presence of ouabain (1 mM) in serosal solution.     

In vitro characterization of calcium transport along the gastrointestinal tract of freshwater rainbow trout Oncorhynchus mykiss

Using an in vitro gut-sac technique, this study examined the mechanisms of calcium (Ca) uptake along the gastrointestinal tract (GIT) of rainbow trout Oncorhynchus mykiss. Ca uptake into three different compartments (mucous-bound, mucosal epithelium and blood space) of four distinct GIT segments (stomach, anterior intestine, mid intestine and posterior intestine) was monitored after luminal exposure to 10 mM Ca saline (radiolabelled with (45) Ca). Ca transport was determined to be both time-dependent and concentration-dependent. The concentration-dependent kinetics of Ca uptake was investigated using varying luminal concentrations of Ca (1, 10, 30, 60 and 100 mM). In the blood-space compartment, Ca uptake was saturable at high Ca concentrations in the mid intestine (suggesting mediated transport), while linear uptake was found in the other gut segments. In the mucous-bound and mucosal epithelium compartments, however, saturation kinetics were found for most GIT segments, also suggesting mediated transport. Manipulation of serosal saline osmotic pressure with mannitol demonstrated that Ca uptake was not greatly affected by solvent drag. Elevated mucosal cadmium (Cd) did not appear to inhibit Ca uptake into the blood space in any of the GIT sections, and Ca uptake did not appear to be sodium dependent. Maximum transport capacities for Ca and Cd were found to be comparable between the gills and gut, but affinities were much higher at the gills (up to 3000 times).     

Chloride channels in the small intestinal cell line IEC-18

Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion.     

Effect of size of Trichinella spiralis (Nematode) infections on glucose and ion transport in the rat intestine

An in vivo perfusion technique, using 3 intestinal loops representing the anterior, mid and posterior regions of the rat small intestine, was used to determine intestinal glucose uptake 5 days after infection with Trichinella spiralis. At high levels of infection (3,000 and 6,000 larvae/rat) net glucose absorption by the intestinal mucosa was significantly impaired in all regions of the small intestine when compared to uninfected controls. At low levels of infection (50 larvae/rat) glucose uptake by the mucosa was significantly enhanced in all 3 regions of the small intestine. Intermediate levels of infections (200-1,000 larvae/rat) also enhanced glucose uptake, but only in the anterior regions of the small intestine. When washings from the small intestine of rats infected with 50 larvae/rat were added to the perfusion fluid used on uninfected rats, glucose uptake was also significantly enhanced. These results suggest that at low levels of infection the intestinal lumen contains a metabolite which may affect the mucosal transport of glucose and the related fluxes of H2O, Na+, Cl-, and K+, in the rat intestine. Luminal [H+] and pCO2 decreased from the proximal to distal regions of the small intestine following perfusion; pO2 was significantly decreased in the proximal and distal regions.     

Parathyroid hormone-related protein regulates intestinal calcium transport in sea bream (Sparus auratus)

Parathyroid hormone-related protein (PTHrP) is a factor associated with normal development and physiology of the nervous, cardiovascular, immune, reproductive, and musculoskeletal systems in higher vertebrates. It also stimulates whole body calcium uptake in sea bream (Sparus auratus) larvae with an estimated 60% coming from intestinal uptake in seawater. The present study investigated the role of PTHrP in the intestinal calcium transport in the sea bream in vitro. Unidirectional mucosal-to-serosal and serosal-to-mucosal 45Ca fluxes were measured in vitro in duodenum, hindgut, and rectum mounted in Ussing chambers. In symmetric conditions with the same saline, bathing apical and basolateral sides of the preparation addition of piscine PTHrP 1-34 (6 nM) to the serosal surface resulted in an increase in mucosal to serosal calcium fluxes in duodenum and hindgut and a reduction in serosal to mucosal in the rectum, indicating that different mechanisms are responsive to PTHrP along the intestine. In control asymmetric conditions, with serosal normal and mucosal bathed with a saline similar in composition to the intestinal fluid, there was a net increase in calcium uptake in all regions. The addition of 6 nM PTHrP 1-34 increased net calcium uptake two- to threefold in all regions. The stimulatory effect of PTHrP on net intestinal calcium absorption is consistent with a hypercalcemic role for the hormone. The results support the view that PTHrP, alone or in conjunction with recently identified PTH-like peptides, counteracts in vivo the hypocalcemic effects of stanniocalcin.     

Vitamin absorption in the in vivo intestine of normal and infected (Hymenolepis diminuta: Cestoda) rats

Uptake and serosal transfer of the vitamins thiamine, riboflavin and folic acid have been studied in vivo in normal and parasitized rats infected with Hymenolepis diminuta (Cestoda). Regional differences in intestinal uptake of all three vitamins in both uninfected and parasitized animals were not satistically significant. In the parasitized intestine mucosal uptake and serosal transfer of thiamine were significantly inhibited, with increased mucosal accumulation of the vitamin as luminal thiamine concentration increased. Apparent increased riboflavin mucosal uptake in parasitized animals, was not matched by the reduced serosal transfer, suggesting adsorption of the vitamin in the unstirred aqueous layers. Mucosal uptake of folic acid increased in the parasitized gut; serosal transfer and mucosal accumulation were not affected. These results, indicating vitamin malabsorption associated with infection by H. diminuta, are consistent with the parasite inhibiting mucosal passive transport mechanisms. This conclusion is supported by the changes in net water fluxes associated with vitamin uptake in the parasitized intestine.     

The role of calcium binding protein in the mechanism of action of cholecalciferol (vitamin D3).

A role has been sought for the calcium binding protein (CaBP) which is synthesised de novo after giving cholecalciferol (CC, vitamin D3) to rachitic chicks. After homogenation of mucosal cells in sucrose media, the CaBP was found in the 78,000 X g supernatant. Therefore, the CaBP is either present in the cytoplasm or in some labile membrane structure, e.g. the microvilli, that is disrupted by homogenation. This intracellular CaBP may facilitate diffusion of Ca into intestinal cells. No secretion of CaBP into the lumen could be detected nor did excess CaBP placed in the lumen increase Ca absorption of rachitic chicks. The mitochondria of duodenal mucosal cells contained most of the Ca being translocated by the small intestine. CaBP caused release of Ca already present in mitochondria and diminished Ca uptake by mitochondria and it appreared to do this by increasing the rate of Ca flux across the mitochondrial membrane. This would explain the greater "turnover" of Ca in mucosal cells of cholecalciferol-treated chicks. These and previous findings have been used to propose a scheme for the effect of cholecalciferol on Ca transport from the small intestine.     

Net fluxes of electrolytes in the rat intestine infected with Moniliformis dubius (Acanthocephala).

The effect of Moniliformis dubius on fluxes of Na+, K+, Cl-, and HCO3-in the rat intestine was determined using a conventional in vivo single-pass perfusion technique. Results for ion and water movements in the uninfected gut were in agreement with previous studies. In the parasitized intestine the jejunal pH was significantly lower than that in control animals, matching the restriction of the parasites to this region of the small intestine. While parasitism did not affect Na+ transport in the distal ileum, Na+ absorption was reduced (pH 7.0), or secretion enhanced (pH 6.0), in the two proximal regions. Cl-absorption was reduced in the distal ileum, but secretion was enhanced in the other two segments. Parasitism also enhanced K+ secretion in all segments. Net H2O absorption was reduced at pH 7.0; at pH 7.0; at pH 6.0 net secretion was also reduced. These changes clearly indicate that a parasite restricted to the jejunum may significantly affect the absorptive and secretory activity of the intestine distal to the site of infection. The results are discussed in the light of current concepts of electrolyte transport. The effect of the parasites on mucosal function distal to their site of attachment is discussed in terms of the release by the parasite of toxin-like substances, changes in the physical-chemical characteristics of the intestinal lumen, and interference with neurohormonal control of gastrointestinal function.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

Calcium transport by basal lateral membrane vesicles from rat small intestine decreases with age

There is a marked decrease in active Ca2+ transport by the rat small intestine with age, particularly between 2 and 12 months. Much evidence suggests that the active component of Ca2+ transport resides in the energy-dependent pumping of Ca2+ across the intestinal basal lateral membrane. Therefore, we have characterized Ca2+ uptake by basal lateral membrane vesicles isolated from young (2-3 month old) and adult (12-14 month old) rats. In vesicles from the proximal duodenum, ATP-dependent Ca2+ uptake was about 4-times greater in the young animal than in the adult. There were no age differences in Ca2+ uptake in the absence of ATP. In vesicles from the ileum, Ca2+ uptake was much less than in the duodenum. The age differences in the ileum were smaller, and ATP-dependent Ca2+ uptake in the young was only twice that seen in the adult. Osmotic lysis of duodenal vesicles reduced Ca2+ uptake to low levels in both age groups, indicating that most of the Ca2+ was being taken up into an osmotically active space. Kinetic studies of Ca2+ uptake showed that there was no change in the apparent affinity but a 5-fold decrease in the Vmax of the adult Ca2+ transport system compared to that of the young animal. This marked decrease in the capacity of basal lateral membrane vesicles to actively transport Ca2+ may contribute to the decline in intestinal Ca2+ absorption with age.     

1-Naphthol beta-D-glucuronide formed intraluminally in rat small intestine mucosa and absorbed into the colon

UDP-glucuronosyltransferase expressed in the rat intestinal epithelial cells is important as the first barrier against chemicals. The distribution of 1-naphthol and its glucuronide formed in rat intestine was estimated by using everted intestine. Roughly 60% of the 1-naphthol added to the mucosal fluid was absorbed into the mucosa of the small intestine and colon within 30 min. Approximately 66% of the 1-naphthol absorbed in the proximal intestine was secreted intraluminally as a glucuronide, and a minimal 9% was transported into the serosal fluid as a glucuronide. In the distal intestine, approximately 34% was secreted intraluminally and 30% was transported into the serosal fluid as a glucuronide. The greatest amount of the glucuronide (37% of the absorbed 1-naphthol) was transported into the serosal fluid, whereas a minimal 7% was secreted intraluminally in the colon. In marked contrast, the colon was found to transport 1-naphthol-glucuronide from the mucosal fluid into the serosal fluid at an approximately 8-fold higher rate than that of the small intestine. These results suggest that, in the small intestine, phenolic xenobiotics are mostly glucuronidated and secreted intraluminally and that the resulting glucuronide is absorbed and transported into the serosal side of the colon.     

Ion activities in the lateral intercellular spaces of gallbladder epithelium transporting at low external osmolarities

The ion activities in the lateral spaces of the unilateral preparation of the gallbladder of Rana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, in mM: 27 Na+, 27 Cl-, 2 K+, 1 Ca++, 4 HCO3-. Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27 mM, a K+ concentration of 1.7 mM, a Ca++ concentration of 0.69 mM and a Cl- concentration of 28.5 mM. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 A diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95 mM, and the concentrations of Na+ and Cl- were both about 5 mM. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.     

Alanine Efflux across the Serosal Border of Turtle Intestine

The exit of alanine across the serosal border of the epithelial cells of turtle intestine was measured by direct and indirect techniques. A decrease or an increase in cell Na did not affect the amino acid flux from cell to serosal solution. Cells loaded with Na and alanine did not exhibit any extrusion of alanine when their serosal membranes were exposed to an Na-free medium containing alanine. However, substantial amino acid extrusion was observed across the mucosal cell border under similar conditions. Although alanine flux across the serosal membrane appeared to be Na-independent, it showed a tendency toward saturation as cellular alanine concentration was elevated. The results are consistent with the postulate that the serosal and mucosal membranes of intestinal cells are asymmetrical with respect to amino acid transport mechanisms. The serosal membrane appears to have an Na-independent carrier-mediated mechanism responsible for alanine transport while transport across the mucosal border involves an Na-dependent process.