Thiocyanate transport across fish intestine (Pleuronectes platessa)

Summary When bathed on both sides with identical chloride-containing salines thein vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107±11 A/cm², a transepithelial potential difference of 5.5±0.6 mV (serosa negative), and a mean mucosal membrane potential of –45.4±0.6 mV. Under these conditions the intracellular chloride activity is 32mm.If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10^–4 m) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue.The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive chloride pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.     

Effect of enzymatic digestion on phenylalanine transport across the small intestine

Phenylalanine accumulation in mucosal strips isolated from rat small intestine was significantly inhibited (P less than 0.01) after preincubation with trypsin, chymotrypsin, phospholipase D and neuraminidase. Unidirectional phenylalanine influx across the small intestine was significantly reduced (P less than 0.01) when the mucosal strips were preincubated with the above mentioned enzymes. Intestinal cell water and volume were not significantly changed (P greater than 0.6) when the intestinal tissues were preincubated with these enzymes.     

Taurine transport across the small intestine of adult and suckling rats

1. Taurine accumulation in intestinal cells of adult and suckling rats reached steady-state after 60 min with an In/Out ratio of 1.46 and 4.66 in the adult and suckling rats respectively. 2. The accumulative capacity of the intestinal strips isolated from suckling rats is almost four times higher than that of adult rats. 3. The steady-state uptake of taurine by the adult and suckling rats intestinal cells is saturable, sodium-dependent and inhibited by ouabain. 4. The calculated Vmax of the mediated component of the steady-state uptake in the suckling rats is three times greater than that of the adult rats, and the affinity is seven fold greater in the suckling as compared to the adult. 5. Taurine influx across the mucosal membrane in the suckling rat is significantly greater than that of the control adult.     

Enzymatic inhibition of lysine transport across the small intestine in vivo

1. Trypsin, at different concentrations, significantly inhibited lysine absorption (P less than 0.05) in a dose-dependent pattern. 2. Maximum inhibition equivalent to 35% below control value was reached with 10 micrograms/ml (100 BAEE units) trypsin with a non-reversible inhibitory effect. 3. Chymotrypsin at 10 micrograms/ml produced a significant decrease (P less than 0.05) of lysine absorption although it did not exceed 5%. Perfusion of both enzymes did not show an additive inhibitory effect. 4. Lysine absorption showed a 39% decrease with 10 micrograms/ml trypsin and 1 X 10(-4) M ouabain, whereas ouabain alone produced 34% inhibition. 5. Lysine absorption showed a 71% decrease with 10 micrograms/ml trypsin in a sodium-free medium, and 70% inhibition with Na-free medium alone. 6. The inhibition of lysine absorption after trypsin treatment could be due to inhibition of the active component of lysine transport.     

Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay

The fluorophore, Phen Green SK (PGSK), was assessed for its suitability to be used in an assay for ferrous ion transport into membrane vesicles. The long wavelengths of excitation and emission (506 and 520 nm, respectively) enable PGSK fluorescence to be detected in membranes, such as the chloroplast inner envelope, that contain high levels of carotenoids which absorb light at lower wavelengths. At low concentrations of Fe2+, less than 3 microM, the interaction between PGSK and Fe2+ appears to result in both static and dynamic quenching of the PGSK fluorescence. The characteristics of this quenching were used to develop a calibration curve to determine the concentration of free Fe2+ at these low concentrations. Pronounced quenching of PGSK fluorescence entrapped within chloroplast inner envelope membrane vesicles was observed when Fe2+ was added. The extent of quenching of PGSK fluorescence trapped inside asolectin vesicles on Fe2+ addition was much less. The kinetics of the quenching of PGSK fluorescence by Fe2+ in vesicles was quite different from that for PGSK and Fe2+ in solution. Using the calibration curve developed for interaction of PGSK and low Fe2+ concentrations the initial rates of iron transport could be determined for the chloroplast inner envelope membranes.     

Effect of somatostatin on D-galactose transport across the small intestine of rats

Somatostatin was found to diminish control and theophylline-treated tissue sugar accumulation as well as control and also to diminish theophylline mucosal to serosal D-galactose fluxes. When Na+ was removed from the bath solution, sugar transport was unaltered by the presence of somatostatin. The same results were obtained with phlorizin in the medium. These results seem to suggest that the action of somatostatin is restricted to the Na+-dependent sugar carrier located on the brush border of the intestinal epithelium.     

Inhibition of sugar active transport across rat intestine in vivo by cadmium

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.     

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

Regulation of ion and water transport across the eel intestine: effects of acetylcholine and serotonin

Summary Both acetylcholine (ACh) and serotonin (5-HT) lowered the serosa-negative transepithelial potential difference (PD) and the short-circuit current (I_sc), accompanied by a decrease in NaCl and water absorption across the eel intestine. These inhibitory effects of ACh and 5-HT were blocked by atropine, a muscarinic receptor antagonist, and ICS-205930, a 5-HT₃ receptor antagonist, respectively. Even after blocking the ACh receptor with atropine, 5-HT inhibited the PD and I_sc, and ACh lowered them after blocking the 5-HT receptor with ICS-205930, indicating that ACh and 5-HT act independently. Similar inhibition in the PD and the I_sc was observed after electrical field stimulation (EFS) which is expected to release endogenous regulators. These effects of EFS were reduced by 70% after simultaneous addition of atropine and ICS-205930. Since atropine and ICS-205930 block ACh and 5-HT receptors, respectively, these results suggest that endogenous ACh and 5-HT are released by EFS.Abbreviations ACh acetylcholine - EFS electrical field stimulation - 5-HT serotonin - I _sc short-circuit current - PD transepithelial potential difference - R _t tissue resistance - TTX tetrodotoxin     

The components of taurine transport across the rat small intestine A kinetic study

Summary The transport of taurine across adult Sprague-Dawley rat small intestine was studied in vitro using small intestinal strips. The kinetics of the transport mechanism were investigated under both steady-state and influx conditions. Our findings were compatible with the presence of two distinct transport mechanisms; a linear non-carrier mediated component and a saturable carrier mediated component, with almost equal contribution from each. The mediated component was found to be largely Na⁺-dependent and exhibited marked inhibition by B-alanine and structurally related sulfur amino acids.     

Action of mercury on sugar transport across rat small intestine, in vivo

Absorption of galactose from in vivo perfused rat jejunum was inhibited by 0.1-0.5 mM Hg2+. A few minutes' delay was required for maximal inhibition values. The effects remained after saline solution washing but were in part reversed by EDTA and in higher proportion by dithioerythritol. Absorption inhibition could be ascribed to impairment of the sugar-Na phlorizin-sensitive cotransport component: The passive apparently diffusional component that remains under 0.5 mM phlorizin and absorption of L-sorbose were unaffected by the metal. Hg action is explained as due to its binding to thiol and perhaps other chemical groups of proteins, at different depths in the membrane, which are directly or indirectly related to the sugar transport system.     

pH-sensitive transport of Fe2+ across purified brush-border membrane from mouse intestine

Recent studies of Fe2+ uptake by mouse proximal intestine brush-border membrane vesicles revealed low-affinity, NaCl-sensitive and high-affinity, NaCl-insensitive, components of uptake (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 381-388). In this study, the former component is demonstrated to show a strong pH dependence with an optimum of pH 6.8-6.9. Studies at pH 6.5, where the low affinity component is inhibited by more than 25-fold compared with pH 7.2, suggest that the pH-sensitive component represents transport across the brush-border membrane followed by intravesicular binding. Cholate extracts of brush-border membrane vesicles contain pH- and NaCl-sensitive Fe2+ binding moieties which may be involved in the transfer of Fe2+ across the intestinal brush-border membrane and subsequent binding inside the vesicles. Fe2+ uptake by brush-border membrane vesicles from the duodenum of hypoxic mice is higher than uptake by vesicles from control-fed animals, suggesting the existence of a regulable brush-border membrane Fe2+ carrier.     

Transamination and asymmetry in glutamate transport across the basolateral membrane of frog small intestine

Transport of L-glutamate across the basolateral membrane of frog small-intestinal epithelium, unlike that of L-alanine, is highly asymmetric; thus the rate constant (K entry) describing the entry of glutamate into the epithelium from the vascular bed across this membrane is one order of magnitude greater than the rate constant (K exit) describing its exit. This asymmetry, which appears not to depend upon the Na gradient, may be important in maintaining a high intracellular concentration of glutamate relative to alanine thereby favouring the production of alanine from glutamate by transamination.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

A radioactive uptake assay to measure ion transport across ion channel-containing liposomes

Here we describe a procedure for incorporating ion channels into lipid vesicles (liposomes) and functional characterization of the channel population by assaying radioactive isotope uptake into these proteoliposomes. The technique as described will work only for potassium channels but can be easily modified, as suggested in the text, for other ion channels and transporters. Purified ion channel proteins in detergent micelles are combined with solubilized lipids. Detergent is subsequently removed from protein-lipid complexes by gel filtration or dialysis into high potassium (high [K+]) buffer. After freezing-thawing and sonication, the resultant larger liposomes are passed over another gel-filtration column to exchange an extraliposomal high [K+] to a low [K+] buffer, thus establishing a large K+ gradient across the liposomal membrane. Trace 86Rb is then added to the extraliposomal space and the reaction begins. If the ion channel is permeable to K+, the K+ inside exits the liposomes down its concentration gradient and the 86Rb outside accumulates in the intraliposomal space until equilibrium is reached. The reaction time course is monitored by measurement of accumulated 86Rb after removal of external 86Rb over an ion-exchange column. The 86Rb flux assay takes 2-5 hours depending on the reaction rate and the number of desired time points.