Analytic validation and comparison of three commercial immunoassays for measurement of plasma atrial/A-type natriuretic peptide concentration in horses.

Measurement of atrial/A-type natriuretic peptide (ANP) concentrations may be of use for assessment of cardiac disease, and reliable data on the analytic performance of available assays are needed. To assess the suitability for clinical use of commercially available ANP assays, intra-assay and inter-assay coefficient of variation and dilution parallelism were calculated for three immunoassays (RIAPen, RIAPhoen, and an ELISAPen) using blood samples from healthy and diseased horses to cover a wide range of ANP concentrations. Further, agreement between assays was assessed using linear regression and Bland-Altman analyses. For all assays, precision was moderate but acceptable and dilution parallelism was good. All assays showed analytic performance similar to other immunoassays used in veterinary medicine. However, the results from the three assays were poorly comparable. Our study highlights the need for an optimised species-specific assay for equine samples.

Plasma atrial natriuretic peptide concentrations in horses with heart disease: a pilot study.

Atrial natriuretic peptide (ANP) is a cardiovascular biomarker that might be useful in assessing the severity of cardiac disease in horses. Plasma ANP concentrations (Cp(ANP)) were compared between horses with heart disease but normal chamber size and function (Group A; n=6), horses with heart disease associated with left atrial (LA) enlargement, LA dysfunction, and/or left ventricular (LV) enlargement (Group B; n=5), and horses with no clinically apparent cardiovascular disease (Group C; n=13). The median (min-max) for Cp(ANP) was significantly higher in Group B (53.5 (36.0-70.7) pg/mL), compared to Group A (12.5 (6.3-19.8) pg/mL) and Group C (13.4 (7.2-34.0) pg/mL). Backwards stepwise multiple linear regression showed that Cp(ANP) in horses with heart disease was related to LA dimensions, but not to LV size, LA function, and LV function. The results indicated that Cp(ANP) in horses might be useful in detecting LA enlargement and that Cp(ANP) could be related to the severity of cardiac disease. Larger prospective studies are necessary to confirm these results.

Fecal shedding of infectious feline leukemia virus and its nucleic acids: a transmission potential.

Although it is assumed that fecal shedding of feline leukemia virus (FeLV) constitutes a transmission potential, no study has been performed showing that feces of infected cats can be a source of infection. In this study, we investigated fecal viral shedding of FeLV and its role in viral pathogenesis with the goal to improve infection control. FeLV RNA and DNA levels were determined in rectal swabs of experimentally infected cats by real-time PCR, and the results were correlated with proviral and viral loads in whole blood and plasma, respectively, and plasma p27 levels. All antigenemic cats shed FeLV RNA and DNA in feces. To determine whether the viral RNA detected was infectious, virus isolation from feces was also performed. Infectious virus was isolated from feces of antigenemic cats, and these results perfectly correlated with the isolation of virus from plasma. Naïve cats exposed to these feces seroconverted, showing that infection through feces took place, but remained negative for the presence of FeLV provirus and p27 in blood, an outcome so far not described. Some of the organs collected after euthanasia were provirus positive at low copy numbers. From these results it is concluded that fecal shedding of FeLV plays a role in transmission, but it is probably of secondary importance in viral pathogenesis. Nevertheless, sharing of litter pans by susceptible and viremic cats could increase the environmental infectious pressure and appropriate measures should be taken to avoid unnecessary viral exposure.

Na-K-ATPase in rat cerebellar granule cells is redox sensitive.

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.

[High intestinal transport activity for nucleosides in cattle: a synopsis]. / Hohe intestinale Transportaktivität für Nucleoside beim Rind: Eine Synopse.

In ruminants large amounts of nucleic acids associated with the microbial cell mass leaving the fore-stomach system via the abomasum enter the small intestine. In dairy cows this amounts to 100-200 g microbial nucleic acids per day. These nucleic acids are very efficiently digested in the small intestine whereby nucleosides were found to be the main degradation products. Therefore, this review centers mainly on the mechanisms of intestinal absorption of nucleosides and their role in nucleic acid digestion. Furthermore, metabolism of nucleosides in intestinal epithelial cells and its bearing for nucleoside absorption and salvage of nucleosides for nucleic acid synthesis in various tissues is considered. According to own studies using isolated intestinal brush-border membrane (BBM) vesicles (BBMV) from dairy cows purine and pyimidine nucleosides are transported actively by two separate Na+ co-transport systems (N1 and N2) across the bovine BBM, whereby transport activity in the small intestine decreases from proximal to distal. Guanosine and inosine appeared to be transported exclusively by N1 while thymidine and cytidine appeared to be transported exclusively by N2. Uridine and adenosine had an affinity to both transporters. In comparison to findings in man and rabbit, transport capacity (Vmax) of N1 and N2 in the BBM of cows was more than 10-fold higher. Similar findings were obtained in BBMV isolated from the small intestine of veal calves with rudimentary forestomach development in regard to nucleoside transport. Therefore, the high intestinal transport activity for nucleosides seems to be a genetically fixed property in the bovine, which is not related to a functional fore-stomach system.

Hypoxic responses of Na+/K+ ATPase in trout hepatocytes.

Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.

Na+ gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles.

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.

Nucleosides are efficiently absorbed by Na(+)-dependent transport across the intestinal brush border membrane in veal calves.

In previous work, a comparatively high capacity for Na(+)-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was observed in dairy cows, which might be related to digestion of the large amount of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na(+)-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amounts of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na(+)-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concentration gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids observed by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids associated with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.

Characteristics of Na(+)-dependent intestinal nucleoside transport in the pig.

Since the capacity of nucleic acid digestion and absorption appears to be comparatively high in the pig, we investigated the properties of transport of (3)H-labelled nucleosides across the porcine intestinal brush border membrane (BBM) using BBM vesicles isolated from the small intestine of slaughter pigs. In the presence of a transmembrane Na(+) gradient, uridine, thymidine and guanosine transiently accumulated in the vesicular lumen beyond the equilibrium (60 min) value suggesting the presence of Na(+)/nucleoside cotransporters in the BBM. The findings of inhibitory studies are consistent with the presence of two Na(+)-dependent nucleoside transporters with overlapping substrate specificity, one for pyrimidine nucleosides (N2) and one for purine nucleosides (N1). Guanosine appeared to be a specific substrate for N1, while this applies to thymidine for N2. Transport of thymidine and guanosine were also inhibited by 2 mmol/l D-glucose and alpha-methyl-D-glucoside. The maximal transport capacity (V(max)) for Na(+)-dependent thymidine and guanosine transport were much higher than reported for other monogastric species. Unlike in other species tested, there was no proximal-to-distal gradient, neither in nucleoside transport activity nor in the inhibition of nucleoside transport by monosaccharides in the porcine small intestine. The high intestinal nucleoside transport activity may contribute to the high digestive capacity for nucleic acids in the pig.

Properties of Na(+)-dependent nucleoside transport in the proximal and distal small intestine of cows.

Large amounts of nucleic acids associated with rumen microorganisms are digested in the proximal part of the small intestine of ruminants. We studied how the proximal-distal gradient in nucleic acid digestion is related to activity of Na(+)-nucleoside transporters in brush border membrane vesicles isolated from the proximal and distal small intestine of cows. Two Na(+)-dependent nucleoside transporters with overlapping substrate specificity were shown to be present at the two intestinal sites, one for pyrimidine nucleosides and one for purine nucleosides. Affinity constants (K(m)-values) for both thymidine and guanosine transport were similar at the two intestinal sites, while transport capacity (V(max)) was 2-3 times higher in the proximal than in the distal small intestine. Glucose and alpha-methyl-D-glucoside (0.1 mmol/l or 2 mmol/l) inhibited transport of thymidine and guanosine markedly only in the proximal small intestine. It is concluded that absorption of nucleosides by the two Na(+)-nucleoside transporters reflects the proximal-distal gradient in nucleic acid digestion.

Active intestinal absorption of nucleosides by Na+-dependent transport across the brush border membrane in cows.

Transport of 3H-labeled nucleosides across the bovine intestinal brush border membrane (BBM) was characterized with BBM vesicles (BBMV) isolated from mid-jejunum of cows because large amounts of nucleic acids are digested in the small intestine of ruminants. Two Na+-dependent electrogenic nucleoside transporters with overlapping substrate specificity were shown to be present in the jejunal BBM, one for pyrimidine nucleosides and one for purine nucleosides. As indicated by inhibitory studies, thymidine seemed to be a specific substrate for the pyrimidine nucleoside transporter, while this applied to guanosine and deoxyguanosine for the purine nucleoside transporter. Uridine and adenosine appear to have an affinity to both transporters. This also applies to deoxyadenosine and deoxyuridine. Nucleobases (uracil, hypoxanthine) did not affect transport of nucleosides. The kinetic constants (Km and Vmax) for Na-dependent thymidine and guanosine transport were 29 and 24 micromol/L and 78 and 51 pmol (mg protein)(-1) s(-1), respectively. These values are much higher than those reported for monogastric species.

Na(+)-dependent transport of D-xylose by bovine intestinal brush border membrane vesicles (BBMV) is inhibited by various pentoses and hexoses.

To detect whether pentoses and hexoses occurring in rumen bacteria or in hemicellulose ingested with feed and partly released in the small intestine have an affinity for the Na(+)-dependent glucose transporter of the bovine intestinal brush border membrane (BBM), we investigated whether these monosaccharides inhibit Na(+)-dependent transport of 14C-labelled D-xylose across the BBM using brush border membrane vesicles (BBMV) isolated from the mid-jejunum of cows. We used D-xylose as the transport substrate, because it has a low affinity for the Na(+)-dependent glucose transporter and thus its uptake into BBMV is more efficiently competitively inhibited by other sugars than that of D-glucose. D-Ribose, D-mannose and L-rhamnose occurring in rumen bacteria significantly inhibited Na(+)-dependent uptake of D-xylose into BBMV, but their inhibitory effect was less than that of D-glucose, D-xylose and phlorizin. This also applied to L-arabinose (and D-arabinose), which is, like D-xylose and D-galactose, a constituent of hemicellulose, and to 2-deoxy-D-glucose. Of all monosaccharides tested, only D-fructose did not affect Na(+)-dependent D-xylose transport. It is concluded that some pentoses and hexoses occurring in rumen bacteria (D-ribose, D-mannose and L-rhamnose) or hemicellulose (L-arabinose and D-xylose) have a low affinity for the Na(+)-dependent glucose transporter of the bovine BBM and may therefore be absorbed from the jejunum when released in the small intestine.

Inhibition of mucosal glycylsarcosine uptake by acetate in rat distal small intestine.

Acetate deriving from microbial fermentation may occur at considerable concentrations in the distal small intestine, where it appears to be absorbed by two different mechanisms: acetate-HCO3- exchange and non-ionic diffusion. Whether acetate affects absorption of other nutrients at this intestinal site is not known. Therefore the influence of acetate (30 mmol l-1) on oligopeptide absorption was studied using an in vitro mucosal uptake technique allowing measurement of substrate uptake across the brush border membrane (BBM). Acetate significantly inhibited mucosal uptake of 14C-labelled glycylsarcosine (Gly-Sar) at pH 6 and pH 7 in the presence of sodium. No inhibition occurred in the absence of sodium. Both acetate and the absence of sodium decreased Vmax of mucosal Gly-Sar uptake without substantially affecting the apparent Km value. Since it is well established that Vmax of peptide transport across the intestinal BBM depends on the size of the transmembrane H+ gradient as a driving force the present findings are in accordance with the assumption that acetate inhibits peptide absorption by attenuating the H+ gradient across the BBM, which depends on the presence of sodium.

H(+)-coupled uphill transport of the dipeptide glycylsarcosine by bovine intestinal brush-border membrane vesicles.

In monogastric species, a considerable portion of amino acid nitrogen is absorbed across the brush-border membrane of the small intestine as small peptides (e.g., tripeptides and dipeptides). In ruminants, however, this process is less clear. Therefore, we investigated the uptake of radioactively labeled glycylsarcosine as a model dipeptide across the intestinal brush-border membrane using brush-border membrane vesicles prepared from the bovine small intestine. Uphill transport of glycylsarcosine was energized by a transmembrane H+ gradient and was further stimulated by an electrical potential difference across the membrane. Transport mediated by a carrier contributes to total glycylsarcosine transport across the brush-border membrane. Comparison of the apparent kinetic constants between brush-border membranes prepared from the proximal jejunum or ileum revealed similar half-saturating substrate concentrations (1.28 and 0.93 mmol/L for proximal jejunum and ileum, respectively), but maximal transport rates appeared to be somewhat higher in the proximal small intestine (2.15 and 1.20 nmol/mg of protein per 3 s for proximal jejunum and ileum, respectively). Uptake of glycylsarcosine was strongly inhibited by other dipeptides, but the amino acids glycine and sarcosine did not affect transport. Inhibition of glycylsarcosine uptake by cephalexin indicated an affinity of the carrier for cephalosporin antibiotics. Transport of intact dipeptides across the brush-border membrane of the small intestine might be of physiological importance in ruminants because the microbial and dietary proteins resistant to rumen degradation are digested and absorbed in the small intestine.

Cinnamate uptake by rat small intestine: transport kinetics and transepithelial transfer.

Due to their ubiquitous occurrence in the plant kingdom, plant phenolics, including monomeric cinnamic acids, are ingested by man and animals in variable amounts with their natural diets. Recently, Na(+)-dependent saturable transport of cinnamic acid across the brush-border membrane of rat jejunum has been described. It was the aim of the present study to characterize this mechanism in more detail. We therefore determined the transport kinetics of mucosal uptake of radioactively labelled cinnamic acid under various conditions using a short-term mucosal uptake technique. In addition, the transfer of cinnamic acid across the jejunal wall was investigated using everted intestinal sacs. Investigations of the kinetics of cinnamic acid uptake by the mid-jejunal mucosa revealed the involvement of two transport components, a diffusive Na(+)-independent mechanism and a saturable Na(+)-dependent mechanism. The results obtained with everted sacs provided further evidence of the existence of an active Na+ gradient-driven transport of cinnamic acid across the intestinal epithelium. In the presence of Na+, a significant accumulation of cinnamate occurred inside the serosal compartment and this was strongly inhibited by serosal ouabain. A decrease in the extracellular pH stimulated mucosal cinnamate uptake by increasing the apparent affinity (1/km). This may be attributable to the involvement of a transmembrane H+ gradient in Na(+)-dependent cinnamate transport because the protonophore FCCP caused a significant reduction of cinnamate uptake only in the presence of Na+. The kinetics of cinnamate transport in the absence or presence of a surplus of either unlabelled cinnamate or unlabelled butyrate indicates a reduction in the apparent affinity of the Na(+)-dependent mechanism involved in cinnamate uptake. These results may be explained by a modification of the mechanism by the intracellular pH. Additionally, competitive inhibition of cinnamate uptake by substances structurally related to cinnamic acid may also be involved.

A Na(+)-dependent mechanism is involved in mucosal uptake of cinnamic acid across the jejunal brush border in rats.

Phenolic acids are present in all plant-derived foods and in most diets. Indirect evidence indicates substantial absorption of phenolic monomers from the gastrointestinal tract. However, the mechanisms involved in the absorptive process are unknown. The present study investigates mucosal uptake of radioactively labeled cinnamic acid as a model substance for monomeric cinnamic acid derivatives (e.g., cinnamic, ferulic or caffeic acid) in the rat jejunum using an in vitro mucosal uptake technique. The results indicate the existence of a Na(+)-dependent saturable transport mechanism for uptake of cinnamic acid across the jejunal brush border membrane. The observed Na+ dependence of jejunal cinnamate uptake seems not to be related to the activity of the Na+,H+ exchanger. Lowering the pH of the incubation medium resulted in a pronounced increase in mucosal cinnamate uptake that can be only partially explained by an increase in nonionic diffusion of cinnamic acid. Furthermore, jejunal uptake of cinnamate seems to be influenced by intracellular HCO3- and/or pH, since the addition of methazolamide to a HCO3(-)- and CO2-free incubation medium significantly inhibited mucosal cinnamate uptake, whereas methazolamide was without an effect in the presence of HCO3- and CO2 in the incubation medium. Unlabeled cinnamic and ferulic acid as well as short-chain fatty acids (acetic, propionic and butyric acid) significantly inhibited Na(+)-dependent uptake of radioactivity labeled cinnamic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of citrate across the brush border and basolateral membrane of rat small intestine.

It was the aim of the present study to investigate the transport of tricarboxylates (citrate, tricarballylate) across the basolateral membrane (BLM) of the small intestine. Experiments were performed using BLM vesicles isolated from the jejunum of rats. For comparison, some experiments with brush border membrane (BBM) vesicles were also performed. Finally, transfer of citrate and tricarballylate across the intestinal wall was investigated using sacs of everted small intestine. Uptake of citrate by BBM vesicles occurs by a Na+ gradient-driven transport mechanism specific for tri- and dicarboxylates. The partially protonated forms of citrate seem to be much better transported than the completely dissociated form, since lowering the extravesicular pH from 7.8 to 5.6 resulted in a marked stimulation of Na(+)-dependent citrate uptake. In contrast to citrate uptake across the BBM, uptake of citrate across the BLM was neither influenced by Na+ nor by pH changes. Neither structurally related tri- and dicarboxylates (tricarballylate, succinate) nor other organic and inorganic anions (e.g. lactate, p-aminohippurate, sulfate, chloride, bicarbonate) significantly influenced citrate uptake by BLM vesicles under cis-conditions. Uptake of citrate as a function of the extravesicular substrate concentration was linear over a concentration range from 0.1 to 10 mmol/l. Thus, citrate uptake under these conditions seems to be Na(+)-independent and not to be mediated by a carrier. However, preloading the BLMV with citrate clearly trans-stimulated the uptake of citrate and tricarballylate, respectively. Furthermore, citrate significantly inhibited tricarballylate uptake into BLMV preloaded with citrate. These results indicate uptake of tricarboxylates across the BLM by an exchange mechanism. Using sacs of everted small intestine, no transfer of intact citrate against a concentration gradient occurred, but some evidence for metabolization of citrate within the intestinal wall was obtained. In contrast, the non-metabolizable tricarboxylate tricarballylate was significantly accumulated in the serosal compartment of everted intestinal sacs.

Intestinal transport of taurocholate in the cat.

In the present study, the transport of taurocholate by brush border membrane vesicles (BBMV), prepared from the proximal and distal half of the small intestine of cats was investigated. Uptake of taurocholate (0.01 mmol/l) into BBMV from the distal small intestine was clearly enhanced in the presence of an inwardly directed initial Na+ gradient compared to choline+ gradient conditions, whereas uptake by BBMV from the proximal half of the small intestine was not substantially different between the two incubation conditions. Calculated uptake of taurocholate at a 10s incubation period was mainly due to transfer of taurocholate into the vesicular lumen rather than to extensive binding of taurocholate to the membranes. Evaluation of the kinetics of taurocholate transport across the brush border membrane of the distal half of the small intestine under Na+ gradient conditions revealed a saturable Na(+)-dependent component and a diffusive component with the subsequent apparent parameters: Vmax (maximal transport velocity) = 0.59 nmol/mg protein.10s, KM (affinity constant) = 0.12 mmol/l, D (diffusion constant) = 2.0 microliters/mg protein.10s. The results indicate Na+/taurocholate co-transport across the brush border membrane of the distal half of the small intestine in cats. Thus, the carrier-mediated intestinal bile salt absorption in the cat appears to be similar as described in other species.

Characterization of the transport of tri- and dicarboxylates by pig intestinal brush-border membrane vesicles.

1. Transport of citrate and fumarate across the pig intestinal brush-border membrane (BBM) was investigated using isolated BBM vesicles. 2. Citrate and fumarate uptake was stimulated by an inwardly directed Na+ gradient consistent with Na+/citrate (fumarate) co-transport. Cis-inhibition and trans-stimulation experiments strongly suggest the existence of a common transport site for tri- and dicarboxylates. 3. The protonated forms of citrate (citrate-1, citrate-2) seem to be much better transported than citrate-3, indicated by the strong stimulation of citrate uptake at an extravesicular pH of 5.6 compared to pH 7.8. 4. Uptake of tri- and dicarboxylates seems to be potential-sensitive since citrate and in particular fumarate transport was enhanced by an inside negative potential difference. 5. Kinetics of succinate transport revealed a single carrier-mediated component with apparent kinetic constants of 0.43 nmol/mg protein-3 s (Vmax) and 0.14 mmol/l (Km).

Transport of tri- and dicarboxylic acids across the intestinal brush border membrane of calves.

Transport of tri- and dicarboxylic acids across the intestinal brush border membrane was investigated using citrate and fumarate as transport substrates. The experiments were performed with brush border membrane vesicles isolated from calf proximal jejunum. Citrate and fumarate uptake by the brush border membrane vesicles occurred by a common Na(+)-dependent transport mechanism that appears to be specific for tri- and dicarboxylates. The protonated forms of citrate (citrate-(1) and citrate-(2] seem to be much better transported than the trivalent form, as indicated by the strong stimulation of citrate uptake at an extravesicular pH of 5.6 compared to that at pH 7.8. Furthermore, citrate transport across the intestinal brush border membrane appears to be mediated by an electroneutral process.