Low-capacity Component Research Articles

N-n-alkylnicotinium analogs, a novel class of nicotinic receptor antagonist: inhibition of S(-)-nicotine-evoked [(3)H]dopamine overflow from superfused rat striatal slices.

The structure of the S(-)-nicotine molecule was modified via N-n-alkylation of the pyridine-N atom to afford a series of N-n-alkylnicotinium iodide salts with carbon chain lengths varying between C(1) and C(12). The ability of these analogs to evoke [(3)H] overflow and inhibit S(-)-nicotine-evoked [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices was determined. At high concentrations, analogs with chain lengths > or =C(6) evoked [(3)H] overflow. Specifically, N-n-decylnicotinium iodide (NDNI; C(10)) evoked significant [(3)H] overflow at 1 microM, and N-n-dodecylnicotinium iodide (NDDNI; C(12)) at 10 microM, whereas N-n-octylnicotinium iodide (NONI; C(8)), N-n-heptylnicotinium iodide (NHpNI; C(7)), and N-n-hexylnicotinium iodide (C(6)) evoked [(3)H] overflow at 100 microM. Thus, intrinsic activity at these concentrations prohibited assessment of inhibitory activity. The most potent N-n-alkylnicotinium analog to inhibit S(-)-nicotine-evoked [(3)H] overflow was NDDNI, with an IC(50) value of 9 nM. NHpNI, NONI, and N-n-nonylnicotinium iodide (C(9)) also inhibited S(-)-nicotine-evoked [(3)H] overflow with IC(50) values of 0.80, 0.62, and 0.21 microM, respectively. In comparison, the competitive neuronal nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-beta-erythroidine, had an IC(50) of 1.6 microM. A significant correlation of N-n-alkyl chain length with analog-induced inhibition was observed, with the exception of NDNI, which was devoid of inhibitory activity. The mechanism of N-n-alkylnicotinium-induced inhibition of the high-affinity, low-capacity component of S(-)-nicotine-evoked [(3)H] overflow was determined via Schild analysis, using the representative analog, NONI. Linear Schild regression and slope not different from unity suggested that NONI competitively interacts with a single nAChR subtype to inhibit S(-)-nicotine-evoked [(3)H]DA release (K(i) value = 80.2 nM). Thus, modification of the S(-)-nicotine molecule converts this agonist into an antagonist at nAChRs, mediating S(-)-nicotine-evoked DA release in striatum.

Kinetic analysis of agonist-induced down-regulation of the beta(2)-adrenergic receptor in BEAS-2B cells reveals high- and low-affinity components.

We examined the interrelationships of internalization and down-regulation of the beta(2)-adrenergic receptor in response to treatment of the BEAS-2B human epithelial cell line with both a series of agonists at high occupancy and with various concentrations of fenoterol that gave occupancies from 0.93 to 0.001. We found that the extent of internalization measured after a 30-min treatment increased as a function of coupling efficiency, with ephedrine, dobutamine, albuterol, fenoterol, and epinephrine giving 0, 7, 17, 48, and 55% internalization, respectively. With the exception of dobutamine, the rates of down-regulation (k(deg)) also showed a dependence on agonist coupling efficiency, giving (in terms of fraction of receptors lost/h) 0.082 with ephedrine, 0.250 with dobutamine, 0.148 with albuterol, 0.194 with fenoterol, and 0.212 with epinephrine. Comparison of down-regulation to internalization showed that weak agonists caused down-regulation in the absence of significant internalization. The extent of internalization caused by fenoterol over a 1000-fold range of occupancy was proportional to agonist occupancy. However, although no internalization was observed with the low concentrations (0.2 and 2 nM fenoterol), these concentrations did cause significant down-regulation. Thus, as with partial agonists, it was clear that down-regulation occurred in the absence of measurable internalization. The kinetics of agonist-induced down-regulation are consistent with a scheme in which down-regulation proceeds by two pathways; a high-affinity, low-capacity component (EC(50) = 0.5 nM) clearly dissociated from internalization and a low-affinity, high-capacity component (EC(50) = 160 nM) closely correlated with internalization.

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K m = 2.2 μM;V max = 23 nmol·(g FW)−1·h−1]; (ii) a low-affinity, high-capacity component [K m = 159 μM;V max = 742 nmol·(g FW)−1·h−1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)−1 h−1];·mM−1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K i = 1–5 μM; and a low affinity (K i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.

Tumour growth results in changes in placental amino acid transport in the rat: a tumour necrosis factor α-mediated effect

The implantation of a fast growing tumour (Yoshida AH-130 ascites hepatoma) to late pregnant rats resulted in no changes in fetal growth, this possibly being associated with an important increase in the fetal uptake of maternal-derived amino acids [Carbó, López-Soriano and Argilés (1995) Endocrinology 136, 3579-3584]. The present investigation was undertaken to see whether the presence of the tumour induced changes in placental transport systems. For alanine transport, although no changes in affinity (Km) were observed, tumour growth resulted in a 192% increase in Vmax in the Na(+)-independent component. Kinetic analysis of the Na(+)-dependent component resulted in two clearly different components: while the low-affinity and high-capacity component was unaffected by tumour growth, the high-affinity, low-capacity component of the tumour-bearing rats showed an important increase in Vmax. (78%). With regard to leucine transport, tumour burden induced important increases in the Na(+)-independent component, not only in Km (262%) but also in Vmax. (189%). Since elevated tumour necrosis factor-alpha (TNF) concentrations have been reported in this kind of tumour model, we performed the same type of transport experiments in rats chronically treated with TNF, the results obtained showing great similarities with those observed with tumour growth. The Vmax. of Na(+)-independent alanine transport was also increased by the cytokine (104%) while no changes were observed in affinity. TNF treatment also induced an increase in the Vmax. (67%) of the Na(+)-dependent (high-affinity, low-capacity) component while no changes in affinity were observed. Concerning leucine kinetics, TNF treatment, as in the case of tumour growth, also increased Km (155%) and Vmax. (72%) associated with Na(+)-independent transport. Interestingly, treatment with the cytokine increased both the Km (43%) and Vmax. (64%) of the Na(+)-dependent component. The inhibition patterns suggest the existence of more that one Na(+)-dependent transport for alanine although the majority of the amino acid is transported through the A system. The results presented suggest that, during gestation, the mother is able to adapt her placental amino acid transport systems to compensate for the nitrogen drainage associated with tumour growth and thus provide the fetus with enough amino acids to allow its normal growth, and that TNF could be responsible for the triggering of this compensatory mechanism.

Comparative survey of blood thyroxine binding proteins in turtles

The nature of plasma thyroxine (T4) binding activity was surveyed in turtles; binding to [125I]T4 was measured on polyacrylamide gel electrophoresis--PAGE--and on minicolumns of Sephadex G-25. An electrophoretically distinct T4 binding protein was identified in all 8 species of Pseudemys studied and in 3 other genera (Chrysemys, Deirochelys, and Emyoidea) of the same family, Emydidae. Levels of this binding activity were highly variable among individuals, but they consistently showed a similar low relative mobility (Rf) compared to albumin, and a relatively low capacity was indicated by displacement with unlabeled T4. Two emydids (Terrapene, Clemmys) showed a similar slow migrating binding peak, but binding activity was low and not as easily displaced by unlabeled T4. T4 binding to albumins was minimal in most of these emydid species, even when binding to the higher affinity, low capacity component was low or displaced by unlabeled T4 (2.5 micrograms/ml). In contrast, there was no clear evidence for a similar high affinity, low capacity binding protein in any of the other 19 species representing 13 genera of 8 families from two suborders. In these species, binding activity on Sephadex G-25 was typically low and binding on PAGE was associated largely with albumin; binding levels for albumins were highly variable. In several nonemydids (from distant lineages), binding activity on Sephadex was elevated and PAGE showed a second binding protein distinct from albumin, but it had high capacity (not readily saturable). Thus, an evolutionary divergence in T4 transport proteins is suggested within Chelonia.

In vitro evidence for the involvement of at least two forms of human liver UDP-glucuronosyltransferase in morphine 3-glucuronidation

Morphine 3-glucuronidation kinetics and the inhibitory effects of a number of xenobiotics on morphine glucuronidation in human liver microsomes have been investigated. In both native and detergent-activated microsomes morphine glucuronidation exhibited biphasic kinetics, with a high affinity, low capacity component and a low affinity, high capacity component. These data suggest the involvement of at least two forms of human liver UDP-glucuronosyltransferase (UDPGT) in morphine glucuronidation. The high affinity morphine-UDPGT activity is likely to be of most importance in morphine glucuronidation in vivo. Chloramphenicol, 4-hydroxybiphenyl, 4-methylumbelliferone, 1-naphthol and 4-nitrophenol were all shown to inhibit the low affinity morphine-UDPGT activity, but only chloramphenicol and 1-naphthol were competitive inhibitors. Each of these xenobiotics were shown to be a non-inhibitor of the high affinity morphine-UDPGT activity, or at least to have considerably lower affinity for this enzyme form(s) than morphine itself. Overall the results provide further evidence for the heterogeneity of human liver UDPGT and, in conjunction with other recent studies (Miners JO et al., Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes, Biochem Pharmacol 37: 665–671, 1988) of the kinetics of human liver glucuronidation reactions, indicate that xenobiotic substrates such as morphine, 4-methylumbelliferone and 1-naphthol may be used to differentiate UDPGT isozyme activities in human liver microsomes.

The chronic effects of desipramine and sertraline on platelet and synaptosomal 5HT uptake in olfactory bulbectomised rats

1. 1. Depressed patients show a characteristic decrease in the rate of uptake of serotonin into their platelets, which is normalised only by clinically effective antidepressant treatment. 2. 2. We also find a decrease in platelet 5HT uptake rates in the olfactory bulbectomised (OB) rat model of depression, which return to normal following three weeks of treatment with desipramine or sertraline. 3. 3. Synaptosomal 5HT uptake appears to consist of a low affinity, high capacity component and a high affinity, low capacity component, both of which are increased in the OB rat, compared to its sham operated control, and normalised by chronic antidepressant treatment. 4. 4. The low affinity uptake of serotonin is not inhibited by in vitro incubation with sertraline, which suggests that the low affinity system may be associated with a non-specific uptake of 5HT into noradrenergic or dopaminergic nerve endings.

Age-related alterations in thyroid hormone binding properties of porcine skeletal muscle cytosol.

Thyroid hormone binding properties were evaluated in cytosol prepared from semitendinosus muscle of 1-d, 3-wk and 5-mo-old Yorkshire-Duroc crossbred pigs. Optimum thyroxine (T4) binding was obtained with 35 ug cytosolic protein in a solution containing 50 mM Tris-HCl buffer, pH 7.4, and 25 mM KCl and 10 mM MgCl2. The half-time of maximum T4 binding was approximately 10 min. Protease digestion of cytosol eliminated specific T4 binding. Muscle cytosol preferentially bound T4 over triiodothyronine (T3). At equimolar concentrations the amount of T3 binding relative to T4 was .1, 50 and 10% at 1 d, 3 wk and 5 mo, respectively. Scatchard analysis of T4 binding revealed a similar high-affinity, low-capacity component in all age groups. However, at 3 wk of age the concentration of the low-affinity, high-capacity binding component for T4 was 12-fold greater than at either 1 d or 5 mo of age. The affinity of this high-capacity binding component did not change with age. The function of thyroid hormones in regulating skeletal muscle growth and metabolism have not been elucidated. These data demonstrate that thyroid hormone binding properties of skeletal muscle do change with age and further research is needed to investigate the possible relationship of these findings to growth regulatory mechanisms.

Thulium binding to the pancreatic beta-cell membrane.

Radioactive thulium (171Tm) was used to probe cation-binding sites in the plasma membrane of beta-cell-rich pancreatic islets microdissected from noninbred ob/ob mice. Temporal studies revealed that 171Tm uptake was rapid, reaching isotopic equilibrium by 60 min. Analysis of the concentration dependence of 171Tm uptake revealed at least two components. At low 171Tm concentrations (0.003--0.18 micrometer), there was a saturable low capacity component, capable of accommodating less than 20 mumol/kg dry weight. At higher 171Tm concentrations (1.0--500 micrometers), a nonsaturable high capacity component capable of binding more than 100 mmol/kg dry weight was observed. 171Tm taken up at 0.18 micrometer exhibited a high degree of mobility. The uptake of 171Tm at this concentration was increased by incubation with chlorpromazine at concentrations known to increase the permeability of the beta-cell plasma membrane. At 0.18 micrometer, exposure to 20 mM D-glucose reduced 171Tm uptake compared with that in islets incubated in the absence of sugar or in the presence of sugars which lack stimulatory effects on insulin release. Such an effect was not observed at 125 micrometers, and none of the sugars influenced the 171Tm uptake of the exocrine pancreas. These data raise the possibility that cation-binding sites in the beta-cell plasma membrane are of physiological significance in the regulation of insulin secretion.

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts.

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.

TRYPTOPHAN TRANSPORT ACROSS THE BLOOD‐BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood‐brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis‐Menten kinetics (normal: Vmax= 19.5 nmol.min−1.g−1. Km= 113 μM; hepatic failure: Vmax, = 33.8 nmol.min−1.g−1, Km= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min−1.g−1; hepatic failure: K= 0.067 ml.min−1.g−1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual Kms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood‐brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood‐brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.

Absence of high-affinity low-capacity components of T 4-binding sites in the cytosol of the bullfrog tadpole

The binding of [ 125I]T 4 to the cytosol of bullfrog tadpole tail tissues was investigated and the binding characteristics were compared with that of [ 125I]T 3, which were reported previously. T 4 binding was slower than T 3 binding and only slightly, if at all, dependent on metal ion. Some deiodination of the hormone occurred during incubation with cytosol at 0°. The ratios of bound T 4 to free T 4 were constant over the concentration range from 6 × 10 −11 to 6 × 10 −8 M, suggesting the absence of high-affinity saturable binding sites for T 4 in the tadpole tail cytosols.