Saturable Transport Mechanism Research Articles

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent K m of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent K m of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a V max of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent Km of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent Km of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a Vmax of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Blood-brain barrier transport rate of quinoline antibacterials evaluated in cerebrovascular endothelial cell cultures

The permeability of the blood-brain barrier (BBB) to ten quinoline antibacterials (gyrase inhibitors) was assessed in an in vitro model consisting of primary cultures of bovine cerebrovascular endothelial cells. The transport kinetics across the cell monolayer was studied over a period of 90 min. The quinolone concentrations were determined by HPLC. The endothelial permeability P e expressing the transport rate differed considerably among individual quinolones, ranging from 0.54·10 −1 (enoxacin) to 0.58·10 −1 (A 60616) cm/min. P e was found to depend on the octanol/buffer partition coefficient (log P) at pH 7.4 as a measure for the lipophilicity of the compounds at physiological pH. The quantitative relationship between P e and log P allows a reliable prediction of the BBB permeability to new quinoline derivatives facilitating the design of quinolones with increased CNS availability. There was neither evidence of the presence of saturable transport mechanisms nor significant metabolism of quinolones at the BBB. The results suggest that quinolones are transported across the BBB by passive diffusion.

A saturable transport mechanism in the intestinal absorption of gabapentin is the underlying cause of the lack of proportionality between increasing dose and drug levels in plasma.

Gabapentin (1-(aminomethyl)cyclohexaneacetic acid) is a neuroprotective agent with antiepileptic properties. The structure is small (molecular weight less than 200), is zwitterionic, and resembles an amino acid with the exception that it does not contain a chiral carbon and the amino group is not alpha to the carboxylate functionality. Gabapentin is not metabolized by humans, and thus, the amount of gabapentin excreted by the renal route represents the fraction of dose absorbed. Clinical trials have reported dose-dependent bioavailabilities ranging from 73.8 +/- 18.3 to 35.7 +/- 18.3% when the dose was increased from 100 to 1600 mg. The permeability of gabapentin in the rat intestinal perfusion system was consistent with carrier-mediated absorption, i.e., a 75 to 80% decrease in permeability when the drug concentration was increased from 0.01 to 50 mM (0.46 +/- 0.05 to 0.12 +/- 0.04). Excellent agreement was obtained between the actual clinical values and the predicted values from in situ results for the fraction of dose absorbed calculated using the theoretically derived correlation, Fabs = 1 - exp(-2Peff) by Amidon et al. (Pharm. Res. 5:651-654, 1988). The permeability values obtained for gabapentin correspond to 67.4 and 30.2% of the dose absorbed at the low and high concentrations, respectively. In the everted rat intestinal ring system, gabapentin shared an inhibition profile similar to that of L-phenylalanine. Characteristics of gabapentin uptake included cross-inhibition with L-Phe, sensitivity to inhibition by L-Leu, stereoselectivity as evidenced by incomplete inhibition by D-Phe, and lack of effect by Gly.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.

Penetration of ceftazidime into bronchial secretions in critically ill patients

Five adult patients admitted to ICU, with respiratory failure, required endotracheal intubation and respiratory support. They were treated with ceftazidime because of lower respiratory tract infections. All patients were given ceftazidime at 1, 2 and 3 g dosages in a randomized sequence and timed serum and bronchial secretion samples collected. Mean peak serum concentrations of 60.3, 148.7 and 224.8 mg/L were found in the three treatment groups and mean trough levels of 1.02, 1.85 and 1.63 mg/L respectively. Ceftazidime appeared rapidly in bronchial secretions reaching mean maximal concentrations of 2.2, 4.81 and 5.69 mg/L in the first sampling period (0-2 h). Serum and bronchial secretions AUCs have been calculated showing that both almost doubled their values between 1 and 2 g dosing, while between 2 and 3 g doses only a moderate and non-significant increase were found. On the basis of these results, the existence of a saturable transport mechanism for ceftazidime from serum to bronchial secretions can be postulated.

Glucose transport system in UMR-106-01 osteoblastic osteosarcoma cells: Regulation by insulin

Insulin is a potent stimulator of collagen synthesis and other osteoblastic cell functions. In various insulin-sensitive tissues, stimulation of glucose transport and glycolytic metabolism are hallmarks of insulin action and may play a role in insulin regulation of cellular function. However, the effects of insulin on glucose metabolism in osteoblast-like cells have not been defined. We therefore characterized 2-deoxy-D-glucose (2-DG) transport in UMR-106-01 rat osteoblastic osteosarcoma cells and examined its regulation by insulin. 2-DG (0.1 mM) uptake was shown to be linear with time over 45 minutes, temperature-sensitive, and inhibited by phloridzin. Competitive inhibition studies against other hexoses demonstrated a transport system stereospecificity for 2-DG similar to that previously demonstrated in fat and muscle cells. Kinetic analysis of 15 minute 2-DG uptake at 25 degrees C demonstrated a saturable transport mechanism with a Km (1.9 mM) similar to that observed for 2-DG transport in other tissues. Insulin stimulated 2-DG transport in a dose-related manner, with significant stimulation observed at 0.5 nM and maximal effect observed at 50 nM insulin. The stimulatory effect of insulin was reversibly inhibited by cytochalasin B (50 microM). Insulin stimulation of 2-DG transport was associated with a 1.7-fold increase in Vmax, while Km remained constant. When insulin effects on glucose transport were inhibited by the addition of 5 mM phloridzin, stimulatory effects on DNA and collagen synthesis were diminished, suggesting that stimulation of glucose transport may play a role in insulin effects on replication and function in osteoblast-like cells.

Slow penetration of thyrotropin-releasing hormone across the blood-brain barrier of an in situ perfused guinea pig brain.

Transport of 3H-labelled thyrotropin-releasing hormone (TRH) across the blood-brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 X 10(-3) to 1.22 X 10(-3) ml min-1 g-1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43-48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood-brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained . L-Prolinamide, the N-terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood-brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood-brain barrier, the rate of transfer being some three times higher than that of D-mannitol.

Glucose transport in osteoblast-enriched bone explants: Characterization and insulin regulation

Insulin has potent effects on osteoblast function both in vivo and in vitro. In various insulin-sensitive tissues, stimulation of glucose transport and metabolism are hallmarks of insulin action, and have been postulated to play a role in insulin regulation of cellular function. However, insulin effects on glucose metabolism in osteoblast-like cells have not been demonstrated. Therefore we examined the in vitro effects of insulin on hexose uptake in an osteoblast-enriched rat bone explant preparation. Uniform 5-mm-diameter punch sections were obtained from the cartilage-free frontal portions of the calvaria of 3-day-old rats, and the periosteum was removed. The resulting sections contained a highly enriched population of osteoblast-like cells as determined by histologic criteria, elimination of calcitonin-stimulatable cAMP generation, and enhancement of PTH-stimulatable cAMP generation per microgram of DNA. Sections were incubated for 24 hr at 37 degrees C in BGJb medium and then transferred to modified glucose-free Krebs-Ringer bicarbonate buffer for 2-deoxy-D-glucose (2-DG) uptake studies. 3H-2-DG uptake was linear with time over 60 min, temperature sensitive, and inhibited by 5 mM phloridzin. Kinetic analysis of 2-DG uptake at 25 degrees C demonstrated a saturable transport mechanism with a Km of 2.2 mM, similar to that observed for 2-DG transport in other tissues. Studies of competitive inhibition by other sugars demonstrated a transport specificity for 2-DG that was comparable to that previously observed in fat and muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptides and the senescent blood-brain barrier

Peptides can influence numerous systems known to be altered by aging. They can affect the passage of non-peptide substances across the blood-brain barrier (BBB) and themselves can cross by both saturable and non-saturable transport mechanisms. The passage of peptides across the BBB is influenced by numerous physiological and pathological events, including aging. Taken together, the evidence suggests that alterations in peptide/BBB interactions may play a significant role in senescence.

Preferential glutamine uptake in rat brain synaptic mitochondria

Glutamine uptake has been studied in purified rat brain mitochondria of synaptic or non-synaptic origin. It was taken up by an active saturable transport mechanism, with an affinity two-times higher in synaptic than in non-synaptic mitochondria ( K m = 0.45 and 0.94 mM, respectively), V max of uptake was 7-times higher in synaptic mitochondria ( V max = 9.2 and 1.3 nmol min per mg protein, respectively). Glutamine transport was found to be inhibited by L-glutamate (IC 50 = 0.64 mM) as well as thiol reagents (mersalyl, N-ethylmaleimide). It is suggested that differential uptake of glutamine in mitochondria of synaptic or nonsynaptic origin may be a major mechanism in the regulation of the synthesis of the neurotransmitter glutamate.

The absorption of piretanide from the gastro-intestinal tract is site-dependent.

The absorption of piretanide was investigated after placing the drug in the stomach, duodenum and ascending colon under visual control. The relative amounts absorbed and the rates of absorption were estimated from the area under the curve and the total mean time, respectively. Similar amounts of piretanide were absorbed and at almost the same rate after placement in the stomach and duodenum; the area under the curve for the stomach was 250 ng h/ml and for the duodenum 243 ng h/ml, the mean absorption times being 1.97 h and 1.51 h respectively. A marked difference was observed in the rate of from the ascending colon; the area amounted to 66 ng h/ml and the mean time to 3.99 h. Although area values for the colon were significantly different from those observed with the stomach and duodenum, it must be emphasised that the amount absorbed depends on the time the drug is in contact with the absorbing surface. There is discussion of whether the differences in absorption between the duodenum and colon can be explained by the physico-chemical properties of the drug alone, or whether the results reflect a saturable transport mechanism.

In Vivo Intestinal Absorption of Selenate and Selenite by Rats

Intestinal absorption of selenate and selenite was investigated in rats by using an in vivo perfusion technique. Different segments of the intestine were perfused with an isotonic solution containing different concentrations of SeO42- or SeO42-. The site of greatest SeO42- absorption was found to be the ileum followed in descending order by the proximal jejunum and large intestine (cecum and colon). Furthermore, SeO42- was absorbed significantly faster from the ileum than SeO42-. The concentration dependence of SeO42- absorption indicates that SeO42- is absorbed by a saturable transport mechanism of the ileal mucosa. Absorption of SeO42- at a concentration of 0.01 mM was not affected by the presence of 1 mM SeO42- in the perfusate. When the SeO42- concentration of the perfusate was increased to 1 mM, the absorptive functions of the ileal epithelium appeared to be generally impaired. It is concluded that selenate is absorbed from the ileum by a carrier-mediated mechanism.

Physicochemical Model for Dose-Dependent DrugAbsorption

A two-tank perfect-mixing tank model was used to simulate GI absorption. The effect of drug parameters (pKa, solubility, and intrinsic wall permeability) and system parameters (pH profile, volume of intestinal contents, and intestinal flow rate) on drug absorption were studied by numerical data simulation. When the dose did not exceed the solubility of the drug in the intestinal lumen, the fraction absorbed depended on the transit rate relative to the absorption rate and the pKa relative to the pH profile, but was independent of drug dose. Saturation of one or both tanks led to dose-dependent absorption. The model was used to simulate absorption of chlorothiazide. Good agreement between simulated and experimental data led to the conclusion that the physical characteristics of chlorothiazide, rather than a saturable transport mechanism at the intestinal wall, may be responsible for the dose-dependent absorption observed for this drug. The model was also used to simulate hydrochlorothiazide absorption. By applying the same system parameters used for chlorothiazide, the model simulation correctly predicted the dose proportionality of hydrochlorothiazide absorption. The lack of dose dependency in this case may be attributed to the higher solubility and pKa of hydrochlorothiazide compared with chlorothiazide.

Lysine transport in the guinea-pig small intestine

Interactions between cationic and neutral amino acids in transport across the brush-border membrane, J mc , of the small intestine have been examined using preparations from the distal rabbit ileum and the rat and guinea-pig mid-small intestine. (1) In the guinea pig, the dependence of J mc Lys on the concentration of lysine is best described in terms of two saturable transport mechanisms in addition to free diffusion. (2) It is shown that the discrepancy between cis-effects of low concentrations of neutral amino acids on the J mc of cationic amino acids, cis-stimulation in the guinea pig contra cis-inhibition in the rabbit and rat, represents species differences. In the guinea pig, imposing sodium-free conditions turns cis-stimulation into cis-inhibition. (3) It is demonstrated that in rat and guinea pig, leucine is transported both by the transport system(s) for cationic amino acids and by transport system(s) which cannot be inhibited by cationic amino acids.

Net transport of glucose from blood to cerebrospinal fluid in the cat

The net transport of glucose from blood to the cerebrospinal fluid compartment of cats was measured by ventriculocisternal perfusion to determine over a large range of serum glucose concentrations the influence of serum glucose levels and their changes on the net transport rate. Changes in serum glucose levels were followed within minutes by corresponding changes in cerebroventricular effluent fluid glucose concentration. At mean values of serum glucose concentration of 6.2 mM and cerebrospinal fluid formation rate of 24.3 μl/min, the net glucose influx rate was 1.6 mmol/min. The effluent fluid-to-serum glucose concentration ratio was 0.25 and decreased when serum glucose was greater than 11.1 mM. The rate of glucose transport from blood to effluent fluid during ventricular perfusion was saturable, and approached a maximum of 3.5 μmol/min at serum glucose levels above 22 mM. From the cererospinal fluid formation and net glucose influx rates the calculated glucose concentration of nascent cererospinal fluid was 6.5 mM and higher than the corresponding serum glucose of 5.6 mM. It is concluded that during perfusion over a wide range of serum glucose concentrations, a saturable mediated glucose transport mechanism can be demonstrated. Changes in serum glucose are rapidly reflected in corresponding effluent fluid glucose levels. From effluent fluid-to-serum glucose concentration ratios and calculations of the glucose in newly formed cerobrospinal fluid, the technique, however, overestimates the glucose influx rates at normal serum glucose levels.

Pharmacokinetic and pharmacodynamic studies of tienilic acid in healthy volunteers.

A simple and reliable HPLC method for the determination of tienilic acid ((TA) Selacryn, Selcryn, Diflurex, Ticrynafen) and its alcoholic metabolite in plasma and urine has been developed. In 8 healthy adult volunteers the plasma and urinary levels of tienilic acid and its alcoholic metabolite, and plasma and urinary levels of sodium, creatinine and uric acid were measured after oral administration of tienilic acid 250 mg. The pharmacokinetic parameters found differed only slightly from those reported in the literature, as there was faster absorption and a shorter half-life. TA is probably excreted by a saturable renal tubular transport mechanism. The pharmacodynamic effects of tienilic acid developed quickly, the uricosuric effect being very impressive and the natriuretic effect moderate. These effects disappeared in about 8 h. An inverse relationship was found between the starting plasma uric acid level in an individual and the maximal uric acid clearance - the higher the plasma uric acid level, the lower was the maximum effect. Plasma tienilic acid level and natriuretic effect were correlative within individuals and intra-individually (p less than 0.05). Urinary tienilic acid level and natriuretic effect were correlated, too (p less than 0.05 to p less than 0.001), but only intraindividually. No correlation between drug level and uricosuric effect was found.

Methodological aspects of studies on the 5-HT uptake mechanism in normal platelets.

The 5-HT uptake mechanism in human platelets was studied in undiluted platelet-rich plasma (PRP). The kinetic data obtained, which were based on 5-HT concentrations ranging from 0.17 to 1.7 mumol/l, were analyzed according to Lineweaver-Burk, Eadie-Hofstee, Scatchard and Sips. In undiluted PRP all the analyses revealed a strict linearity between 5-HT concentrations and initial uptake velocities. Only one site for active transport seemed to be involved and no passive diffusion was present. The apparent Km and Vmax in a 95 per cent confidence interval (n = 25) were 1.20 +/- 0.14 mumol/l and 1.40 +/- 0.15 pmol/10(6) platelets and minutes, respectively. Dilution of PRP with autologous platelet-poor plasma or Ringer's solution markedly influenced the active 5-HT transport mechanism. The addition of minute amounts of supernatant from lysed autologous platelets to PRP totally impaired the active 5-HT uptake. It is concluded that relevant studies on the active 5-HT transport can be performed only in fresh undiluted PRP. The commonly used Lineweaver-Burk analysis does not discriminate sufficiently between fully saturable and non-saturable transport mechanism and therefore cannot be used for the detection of irregularities in the 5-HT transport.

Maternal-Fetal Transfer of Ascorbic Acid in the Guinea Pig

Placental transfer of ascorbic acid was studied in the guinea pig using an in situ placental perfusion technique. A total of 14 animals were studied during their last week of gestation. The fetus was shown to have higher plasma total ascorbic acid (TAA) levels than the mother prior to maternal ascorbic acid infusion. However, following maternal venous infusion this normal gradient for TAA (fetal greater than maternal) disappeared and a new gradient was established (maternal greater than fetal). As maternal plasma TAA levels rose, the level of TAA in the placental perfusate steadily increased to a maximal concentration of approximately 0.85 mg/dl. This occurred when maternal plasma TAA levels were above 3.0 mg/dl. These results demonstrate that a saturable or carrier-mediated transport mechanism is involved in the placental transfer of ascorbic acid in this species. The apparent Vmax (the maximal transport rate by the saturable system per gram placenta) and the Kt (or one-half saturation concentration) were estimated to be 8.3 nmoles/minute and 0.12 mM, respectively. Additional results are presented to suggest that dehydroascorbic acid (DHA) may be the predominant form of the vitamin crossing the guinea pig placenta.

Transport of hypoxanthine by human diploid skin fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase

Summary Transport of purine bases and nucleosides by a variety of mammalian cell lines is generally accomplished by facilitated diffusion, a non-concentrative, saturable process. However, previous investigators have been unable to detect a saturable component for the transport of hypoxanthine by human fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase, implying that in normal cells the enzyme actively participates in transport. In the present study we have used the phenomenon of countertransport to demonstrate the existence of a saturable transport mechanism in hypoxanthine-guanine phosphoribosyltransferase-deficient human diploid skin fibroblasts.