Effect of anaerobiosis, dinitrophenol and fluoride on the active intestinal transport of galactose in snail.

The active transport of galactose across the intestinal wall (everted sacs) of the snail Cryptomphalus hortensis Müller has been studied in vitro, under several metabolic conditions. Anaerobiosis does not change the serosal/mucosal galactose gradients which are developed in oxygen atmosphere. Dinitrophenol (10(-4) M) greatly increased the O2 uptake by the tissue and clearly inhibits the sugar transport. At 5 times 10(-4) M concentration, DNP totally prevents the uphill transport while the O2 uptake is normal. The inhibition produced by DNP does not increase by anaerobiosis. Fluoride inhibits the galactose transport and also the O2 uptake. It is deduced that in snail intestine the energy for the active transport of galactose can be supplied by aerobic as much as by anaerobic metabolism. The inhibition by dinitrophenol seems to be independent of its uncoupling action on the oxidative phosphorylation. The inhibitory effect of NaF may be due both to glycolisis inhibition and to alteration of the digestive epithelium.     

Influence of aerobic energy deficit, ouabain and harmaline on the phenylalanine and galactose active transport by snail intestine

The effect of anaerobiosis (N2 bubbling of the medium) or 10(-4) M dinitrophenol on the penetration of 0.5 mM Phe in snail and rat everted intestine, in 2 min and 30 min incubation periods, has been studied. The aerobic energy deficit inhibits the amino acid net entry in both species, but whereas the active transport is annulled in rat, snail intestine is capable of continuing to accumulate Phe against a gradient. The prolonged action (30 min of preincubation) of 1 mM ouabain inhibits 0.1 mM Phe and 0.1 mM galactose entry in snail intestine. Amino acid uptake is far higher than the one obtained in the absence of Na+, in which condition Phe keeps accumulating against a gradient in the tissue water. Galactose active transport, instead, becomes null in the presence of the glucoside or in the absence of Na+. One mM harmaline is able to inhibit the initial entry of galactose into the tissue, while higher than 5 mM concentrations are required to inhibit that of Phe. Results confirm that snail intestine is capable of easily carrying out active transport processes with energy from anaerobic origin. On the other hand Phe transport is less sensitive to the absence of Na+, presence of ouabain or harmaline than that of galactose, so that contrary to what has been observed for the sugar, the active transport of the amino acid is not annulled in any of the three conditions.     

IC50 of trifluoperazine and RMI 12330A for the theophylline and phloretin-induced increase in intestinal D-galactose accumulation

The effect of trifluoperazine and RMI 12330A on D-Galactose accumulation was studied in isolated rat intestinal mucosa. Both drugs inhibited the theophylline and phloretin-induced increase in tissue sugar accumulation in a concentration-dependent fashion, with IC50 values close to 10(-6) M. These findings suggest that calmodulin might mediate the theophylline and phloretin actions on galactose transport in intact rat ileum.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.     

Inotropic effects and changes in sodium and calcium contents associated with inhibition of monovalent cation active transport by ouabain in cultured myocardial cells

Cultured monolayers of spontaneously contracting chick embryo ventricular cells were perfused with culture medium containing ouabain. Contractile state was monitored by an optical-video system recording amplitude and velocity of cell wall motion. Positive inotropic effects of 2.5 x 10(-7) to 10(-6) M ouabain were manifest within 1.5-2 min, and reached a stable plateau within 5-6 min. The inotropic effect was fully reversed within 5 min after washout of ouabain. Inhibition of uptake of 42K+ (or the K+ analog 86Rb+) and efflux of 24Na+ occurred 1.5-2 min after exposure to ouabain. The degree of inhibition of transport was closely related to the magnitude of the positive inotropic effect throughout the ouabain concentration range 10(-7) to 10(-6) M. After washout of ouabain from monolayers, the monovalent cation active transport rate returned to normal within 1 min. Thus, both the onset and offset of inotropic action of ouabain were closely related temporally to inhibition of the sodium pump. Exposure to ouabain caused significant increases in exchangeable Na and Ca contents that appeared to be developed within 5 min. These data support the hypothesis that inhibition of monovalent cation active transport by ouabain is causally related to the development of positive inotropy and are consistent with modulation of Ca content by intracellular Na+ via the Na+-Ca2+ exchange carrier mechanism.     

Cadmium stimulation of Na-independent organic acid transport in the kidney tubules

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.     

Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Effects of the fraction (1-10 kDa) of the brain of a Yakut horse on kinetic parameters of Ca2+ transport system in sarcolemma vesicles of cardiomyocytes]

The effect of the fraction (1-10 kDa) obtained from the brain of cold-adapted animal (Yakut horse) on Ca2+ transport in sarcolemma vesicles of cardiomyocytes was investigated. It was shown that during insertion of Yakut horse brain fraction into incubation medium at the concentration from 10(-9) M to 3.10(-5) M at Ca2+ transport substrate concentration from 0.1 mM to 1.0 mM, the rate of Ca2+ passive penetration into vesicles slightly increased and at Ca2+ transport substrate concentration 3 mM, which is physiologic, a decrease of rate values was established for all concentrations of the fraction (1-10 kDa) of Yakut horse. While studying the kinetics of an active Ca2+ transport for all investigated concentrations of the fraction (1-10 kDa) of Yakut horse brain from 10(-9) M to 3.10(-5) M at Ca2+ concentration in incubation medium from 10(-7) to 3.10(-6) M, calcium accumulation rates by vesicles exceeded control values. So we can suppose that application of brain fraction (1-10 kDa) of genotypically cold-adapted animal, results in a decrease of intracellular Ca2+ concentration.     

Prostaglandin D2 diminishes transmucosal potential difference in rat colonic mucosa in vitro in contrast to the increasing effect of prostaglandin E1

The effect of Prostaglandin D2 (PGD2) on ion transport was investigated in the rat colon in vitro. Ion transport across the intestinal mucosa was estimated by transmucosal potential difference (PD) and short circuit current (Isc) in the Ussing chamber. PGD2 added to the serosal reservoir induced a sustained reduction in PD and Isc at the concentration of higher than 10(-7)M, producing the maximal decrease at 10(-5)M. PGD2 at 10(-5)M completely blocked the increase in PD elicited by prostaglandin E1 (PGE1), theophylline, dibutyryl cAMP or serotonin. Adenylate cyclase activity was determined in the colonic mucosal homogenates after addition of PGD2 and PGE1. Treatment with PGD2 or PGE1 caused a significant increase in the enzyme activity. Combined treatment with both prostaglandins induced no more increase than that elicited by PGE1 alone. These results suggest that PGD2 has an anti-secretory effect on the rat colon and it may regulate the ion transport process through other mechanism than the modification of cyclic AMP concentration in mucosal cells.     

Energetic cooperation via ion-permeable junctions in mixed animal cell cultures

Low ouabain concentration (1 x 10(-6) M) is shown to decrease intracellular K+ (K+in) and to increase intracellular Na+ (Na+in) in human fibroblast cell cultures. The same ouabain concentration was without effect upon K+in ad Na+in in rodent cultures such as BHK-21, mouse fibroblasts and rat glyoma C6 cells. K+in and Na+in in the mixed cultures of human and BHK-21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 x 10(-6) M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain-sensitive. The gap-junction-mediated dye transfer was revealed between human and BHK-21 cells. Such an effect was very small in the human-C6 cell mixed culture. It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Effect of nitrogen and oxygen active compounds on exchange of Ca2+ and H+ through the myometrial cell membrane

While using myometrium sarcolemma vesicles the action of sodium nitroprusside, NO2-, NO3- and H+ on delta pH-dependent Ca(2+)-transport and passive permeability for H+ vesicles sarcolemma was estimated in the wide concentration range (10(-10)-10(-3) M) of the substances tested. In order of studying calcium transport 45Ca2+ was used, while for H+ translocation registrating via sarcolemma delta pH-indicator 14C-methylamine was applied. Sodium nitroprusside was displayed as weakly effective, while nitrite-anions essentially increased delta pH-dependent Ca2+ transport in the physiologically significant nanomolar concentration region, however in the micromolar region these substances effect failed to differ from the control and restored its intensity starting at 10(-4) M and more. Under the experiment sodium nitroprusside produces considerable quantities of NO2-. Effectory action of NO3- was similar as of NO2-. In the micromolar region the compounds estimated increased considerably sarcolemma passive permeability for H+. Hydrogen peroxide decreased delta pH-dependent Ca(2+)-transport by 10(-8) M and 10(-3) M while at the concentration equal to 10(-3) M increased the sarcolemma passive permeability for proton. Sodium nitroprusside and NO2(-)-effect on the vesicles passive permeability for proton failed to be prevented by dithiotriitol, while H2O2 action was completely removed. The conclusion about the complex concentration-dependent character of the active oxygen metabolities to the sarcolemma transport processes was made, and it's noticeable that the important role in vivo, probably could be played by NO (NO2-) stable nitric metabolities.     

Glucagon binding by isolated chick hepatocytes

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.     

D-galactose uptake by snail intestine: competitive inhibition by phlorizin and sodium dependence

The net entry of galactose into the tissue of snail everted intestinal rings with 2 or 15 minute long incubation periods has been measured. With 10(-4) M phlorizin, the mediated transport is completely blocked while only the passive entry of sugar is produced. Lower concentrations of the glycoside partially inhibit transport according to competitive inhibition kinetics (K1 = 10(-7) M). The transport of galactose is Na+ dependent. In the absence of Na+, transport ceases and the sugar entry can be explained through simple diffusion. With 15 mM Na+ (control 71,4 mM) transport diminishes and a marked increase in the apparent Km with no changes in the Vmax is observed. One mM harmaline completely blocks galactose (0.5 mM) transport. One mM ouabain also makes transport null, but only after tissue preincubation with the inhibitor on the serosal side.     

Inhibition of intestinal sugar transport by phenformin.

The effect of phenformin on the absorption of D-glucose and D-galactose by hamster and rat intestine, was studied. Phenformin did not affect D-glucose absorption by rat intestine, but it inhibited at 10(-3) to 10(-2) M the absorption of D-glucose and D-galactose by hamster intestine. The inhibition was higher when D-glucose was tested. Phenformin also inhibited active accumulation of these sugars by rings of hamster small intestine, in vitro; this effect was greater when D-glucose was utilized. The drug inhibits the oxygen uptake in the tissue in the absence or in the presence of added substrate. Phenformin, as previously suggested, does not seem to act as a specific inhibitor on D-glucose transport, but most likely by its inhibitory effect on mitochondrial respiration.     

Blood-Brain Transfer of Galactose in Experimental Galactosemia, with Special Reference to the Competitive Interaction Between Galactose and Glucose

The interaction between glucose and galactose during transport across the cerebral capillary endothelium was studied in anesthetized rats. Although galactose is present in the diet of suckling mammals and is a potential substrate for brain metabolism in adult mammals, its effect on glucose transport in adult rats is unknown. A kinetic model was formulated to analyze the effect of chronically elevated galactose levels on glucose transport in adult rats. The analysis indicated that galactose and glucose compete for the same transport mechanism in the cerebral capillary endothelium. The Tmax of glucose and galactose were both about 380 mumol 100 g-1 min-1 and the Kt of galactose (30 mM) was about three times that of glucose (10 mM). During prolonged galactosemia in adult rats, neither the Tmax, nor the Kt of either competitor changed substantially when compared with rats subjected to acute galactosemia. At 10 mM galactose in plasma in rats with acute galactosemia, the inhibition of glucose transport, simulated a 25% reduction of plasma glucose, and in rats with chronic galactosemia a 20% reduction. This moderate effect is in contrast to the effect of galactose in suckling rats in which 10 mM galactose in plasma reduced the glucose transport to a level corresponding to a 50% reduction of the plasma glucose concentration.     

Carbohydrate transport in Moniliformis dubius (Acanthocephala). I. The kinetics and specificity of hexose absorption.

The uptakes of 14C-glucose, -2-deoxyglucose, -mannose, -N-acetylglucosamine, -3-0-methylglucose, -fructose, and -galactose by female Moniliformis dubius were nonlinear, saturable functions of hexose concentration. Kinetic and inhibition studies indicated that glucose and 2-deoxyglucose were absorbed via a single common transport locus. Mannose, N-acetylglucosamine, 3-0-methylglucose, fructose, and galactose (in decreasing order of effectiveness) inhibited the uptake of glucose in a completely competitive manner; their absorptions appeared to be mediated by the glucose transport locus and, to some degree, by one or more additional transport systems. Kinetic studies suggested that the apparent inhibitions of 14C-glucose uptake by maltose and glucose-6-phosphate were due to free glucose liberated through the action of surface hydrolases. The uptake of 14C-glucose was also inhibited by salicin, alpha-methylglucoside, and beta-methylglucoside, but not by pentoses, L-hexoses, sugar alcohols, disaccharides (except maltose), gluconic acid, glucuronic acid, phlorizin, or ouabain. Glucose uptake was not Na+-dependent.     

Inhibition of the intestinal transport of uracil by hexoses and amino acids

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.