Low-affinity Process Research Articles

Biochemical evidence for cholinergic involvement in the Limulus brain

1. The transport of [ 3H]choline by the corpora pedunculata of the circumoesophageal ring gland (brain) of Limulus polyphemus was studied. Corpora pedunculata slices were incubated individually in Chao's solution containing 0.01 μM [ 3H]choline at room temperature (25 ± 2°C) and readily accumulated the radiolabel from the extracellular environment. 2. The corpora pedunculata uptake of [ 3H]choline was linear over 60 min. 3. The kinetic analysis indicated the existence of dual uptake systems for choline within the corpora pedunculata, a high affinity choline uptake process ( K m = 0.54 μM and V max = 0.037 pmoles/mg/min) and a low affinity process ( K m = 137 μM and V max = 6.3 pmoles/mg/min). 4. The high affinity choline transport system was dependent on sodium ions and was inhibited by micromolar concentrations of hemicholinium-3. 5. The pre-exposure of the corpora pedunculata to Chao's solution containing 90 mM potassium for 15 min resulted in a 24% increase in the velocity of the high affinity choline uptake process ( V max = 0.046 pmoles/mg/min). 6. The 90 mM potassium Chao's pretreatment stimulated a substantial increase in the synthesis of [ 3H]acetyleholine by the corpora pedunculata. 7. The results suggest that the high affinity choline uptake process within the Limulus corpora pedunculata is associated with the synthesis of the transmitter acetylcholine, presumably within cholinergic terminals in this tissue.

High-uptake and low-uptake forms of proteoglycans secreted by arterial smooth muscle cells.

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 X 10(3) and 180 X 10(3) and are characterized as chondroitin-sulfate-rich (A) and dermatan-sulfate-rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis. Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 microM) one cell may endocytose up to 1.5 X 10(6) proteoglycan B molecules/h. Proteoglycan A is internalized at a 10-fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 microM). Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A. The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high-affinity receptor-mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low-affinity endocytotic processes.

Acute effects of cholinesterase inhibitors on uptake of choline in the rat iris

The choline uptake system has been characterized in the albino rat iris. The isolated iris of the rat accumulates choline (Ch) by a high affinity ( K m = 3.23 μM) and a low affinity ( K m = 68.4 μM) process. The uptake of 1 μM Ch at 37°C was inhibited in a dose-dependent manner by hemicholinium (IC 50 is 70 μM) and by ouabain (IC 50 is 1 mM). The high affinity uptake was temperature and sodium-dependent. At 0°C uptake of Ch was reduced by over 85% and it was not sensitive to hemicholinium. This suggests that choline is taken up by a high affinity active transport into the cholinergic terminals of the iris. The uptake of 1 μM Ch at 37°C was inhibited by 1 mM scopolamine and was reduced in a dose-dependent manner by the irreversible anticholinesterase diisopropylfluorophosphate (DFP). Physostigmine, an anti-cholinesterase lacking agonistic action, decreased the uptake of Ch to 73% of control at 1 mM. The rat iris is a much more homogeneous and readily accessible tissue than the CNS, therefore, it offers several advantages in pharmacological studies of drug action on a selective population of cholinergic terminals.

Some properties of the thiamine uptake system in isolated rat hepatocytes

A Kinetic study of [ 14C]thiamine uptake over a concentration range from 0.1 μM to 4mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a K t of 34.1 μM and V max of 20.8 pmol/10 5 cells per 30 s and a high affinity process with a K t of 1.26 μM and V max of 1.21 pmol/10 5 cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K + medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na + and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a K t of 40.5 μM, which was similar to the K t value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rate of thiamine by pyrophosphorylation at physiological concentrations of thiamine.

Catecholamines in the coelenterate Renilla k�llikeri

The characteristics of uptake of 3H-noradrenaline (3H-NA) and 3H-adrenaline (3H-A) in the tissues of the sea pansy, Renilla kollikeri, were studied by in vivo incubations. Lineweaver-Burk plots indicated two components of catecholamine accumulation, one representing a high-affinity uptake with an apparent Km of 4.91×10-7 M (3H-NA) or 4.39×10-7 M (3H-A), and the other a low affinity process with an apparent Km of 5.52×10-5 M (3H-NA) or 1.49×10-5 M (3H-A). The high-affinity uptake of both tracers was strongly inhibited at low temperature and in a calcium-free medium, thus suggesting the involvement of a carrier-mediated transport mechanism, but was largely insensitive to sodium omission and ouabain. Accumulations of 3H-NA, but not 3H-A, were highly desipramine-sensitive.

Imipramine does not act as a false transmitter in the rat hypothalamus

[ 3H]Imipramine was accumulated by rat hypothalamic slices mostly by a passive low-affinity process. Accumulated [ 3H]imipramine was released by 60 mM potassium, but the release was small and was not Ca 2+-dependent. Imipramine induced some release of [ 3H]norepinephrine. None of these events were abolished by pretreatment of the rats with reserpine. These results do not favor the view that imipramine acts as a false transmitter in the rat hypothalamus. Rather, imipramine appears to be accumulated in an extragranular pool from which it releases extragranular norepinephrine.

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37°C for 48 h with [ 14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4–20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day −1 and 1.70 day −1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.

Active transport of amino acids in Thiobacillus thioparus is a low-affinity process.

A method for the isolation of amino acid auxotrophs of Thiobacillus thioparus is described. Characterization of a leucine auxotroph indicated that leucine biosynthesis in T. thioparus was not different from that of heterotrophic bacteria. T. thioparus cells accumulated amino acids via an active mechanism. Kt values of amino acid transport were between 15 and 330 microM, and Vmax values were 200 to 350 pmol min-1 mg of protein-1. Amino acid transport was carried out by a limited number of systems, each responsible for the uptake of several amino acids. Amino acid auxotrophs of T. thioparus exhibited transport and growth properties similar to those of transport-deficient mutants of heterotrophs which lost the high-affinity, but retained the low-affinity, amino acid transport systems.

The formation of carbamazepine epoxide by rat liver microsomes: an investigation of the biphasic kinetic profile

A biphasic profile has been found for the rat hepatic microsomal formation of carbamazepine 10, 11 -epoxide from varying concentrations of carbamazepine (CBZ). The two optima for epoxide formation appeared at substrate concentrations of about 0.3 mM and 1.0 mM CBZ, respectively, with a nadir occuring at 0.4 – 0.6 mM CBZ. The biphasic nature of the velocity-substrate profile was not due to metabolism or disappearance of the epoxide. Pretreatment of rats with phenobarbital or CBZ produced an increase in the epoxide formation at both low and high CBZ concentrations, whereas phenytoin (DPH) pretreatment increased epoxide only at low CBZ concentrations. 3-Methylcholanthrene treatment did not increase epoxide formation at either low or high CBZ concentrations. High and low affinity processes for epoxide formation developed in parallel in young rats. DPH added in vitro inhibited only the epoxide formation at high CBZ concentrations. This inhibitory effect increased with age of the rats. These findings indicate that CBZ 10, 11 -epoxide formation in rat liver microsomes proceeds by two metabolic pathways distinguished by substrate affinity and inhibition. Analysis of data from previous clinical studies reveals a biphasic pattern for plasma levels of CBZ, and its 10, 11 -epoxide.

Glutamate receptor binding to cat central nervous system membranes.

L-[3H]Glutamate exhibited specific binding to fresh membranes of cat CNS under physiological conditions of pH and temperature. This binding occurred in the absence of sodium ions. Kinetic analysis of the data for cerebellum suggested the presence of two distinct binding sites: a high-affinity process (Kd = 0.33 microM) with a capacity of 15 pmol/mg protein and a low-affinity process (Kd = 1.8 microM) which had a capacity of 65 pmol/mg protein. Several structural analogues of glutamic acid were able to appreciably inhibit the binding of [3H]glutamate. The distribution of glutamate binding between 12 regions of the CNS was measured. The amygdaloid complex exhibited the highest binding followed by hippocampus > hypothalamus identical to visual cortex identical to thalamus identical to caudate nucleus > olfactory bulb identical to tectum identical to cerebellum > dorsal pons identical to medulla > cervical spinal cord. These findings are consistent with the binding of [3H]glutamate being to its receptor.

Bicuculline-sensitive γ-aminobutyrate binding processes in a synaptosome-enriched fraction of rat cerebral cortex

The binding of low concentrations of [ 3H]y-aminobutyrate (GABA) to a synaptosomal fraction of rat cerebral, cortex was studied in the presence and absence of added Na +; unlabelled GABA and bicuculline methiodide were used to estimate ‘GABA-sensitive’ and ‘bicuculline-sensitive’ binding sites. Significant amounts of [ 3H]GABA were bound to the particles in both the presence and absence of added Na +. In Na +-free medium, a single bicuculline-sensitive, high-affinity [ 3H]GABA binding process was detected, its GABA-sensitive sites having K B ∼- 10 −5 M. In Na +-containing medium, two bicuculline-sensitive, high-affinity [ 3H]GABA binding processes were detected, the lower-affinity process having GABA-sensitive sites with K B ∼- 10 −5 M and the higher-affinity process having GABA-sensitive sites with K B ∼- 4 × 10 −8 M Our findings suggest that bicuculline-sensitive components of [ 3H]GABA binding in the absence of Na + and in the presence of Na + (higher-affinity process) could represent estimates of Na +-indepen dent and Na +-dependent synaptic GABA-receptors. The bicuculline-sensitive component of lower-affinity [ 3H]GABA binding in the presence of Na + might be related to an action of bicuculline on GABA transport. Two GABA-receptor agonists displaced about the same amount of [ +H]GABA as did bicuculline in Na +-free medium, indicating further that a synaptic GABA-receptor was involved in [ 3H]GABA binding under these conditions. Nipecotic acid markedly inhibited [ 3H]GABA binding in both the presence and absence of added Na +, indicating that some transport sites become occupied by GABA in the absence of added Na + and that nipecotic acid might interact to some extent with synaptic GABA-receptors.

Effect of nisoxetine on uptake of catecholamines in synaptosomes isolated from discrete regions of rat brain

Nisoxetine inhibits the high affinity uptake of norepinephrine by synaptosomes hypothalamus, brain stem and cerebral cortex of rat brain with inhibitor constants, K i values, of 1, 2 and 6 nM respectively. The drug also inhibits the high affinity uptake of dopamine by hypothalamic synaptosomes with a K i value of 9 nM. In corpus striatum, however, the K i values for nisoxetine to inhibit the uptake ofdopamine and norepinephrine are 360 and 220 nM respectively. Thus, nisoxetine has about 200-fold greater affinity for the uptake sites of norepinephrine than for those of dopamine. Nisoxetine administered at 10 mg/kg i.p. to rats reduces the high affinity uptake of catecholamines by synaptosomes of brain regions. A low affinity process for uptake of catecholamines also occurs in synaptosomes of hypothalamus, brain stem and cerebral cortex but is not blocked by 10 −8M nisoxetine. The significance of the low affinity uptake processes is discussed.

The uptake of [3H] choline by snail (Helix pomatia) nervous tissue in vitro.

Abstract— When suboesophageal ganglia of the snail Helix comalia were incubated at 25°C in a medium containing [3H]choline, tissue: medium ratios of about 14:1 were obtained after 20 min incubation, and only 15°, of the accumulated choline was metabolized to form [3H]acetylcholine. The uptake of [3H]choline showed saturation kinetics and was dependent upon temperature and sodium ions. Kinetic analysis suggested the existence of a high affinity uptake process (Km= 1.7 μM, Vmax= 0.21 nmol/g/min) and a low affinity process (Km= 100 μM, Vmax= 1.2 nmol/g/min). The high affinity uptake differed from the low affinity system in that it was sensitive to various metabolic inhibitors and was competitively inhibited by low concentrations of hemicholinium‐ and acetylcholine. Neither uptake system was greatly influenced by the absence of calcium, potassium or magnesium ions or by the presence of low concentrations of 5‐HT, dopamine. tetrabenazine, chlorpromazine, decamethonium, nalaxone or imipramine. The high affinity uptake process may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.

The uptake of biogenic monoamines by the isolated auricle of the snail (Helix pomatia).

—The uptake of [3H]5HT, [3H]dopamine, [3H]noradrenaline and [3H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [3H]5-HT, [3H]dopamine, [3H]octopamine or [3H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a Km1 value of 6.0 ± 10−8m and a Vm1 value of 0.115 nmol/g/min while the lower affinity process had a Km2 value of 1.04 ± 10−6m and a Vm2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a Km value of 5.02 ± 10−8m and a Vm value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for Km and Vm of 1.55 ± 10−7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.

The in vitro uptake of biogenic amines by snail (Helix pomatia) nervous tissue

The uptake of [ 3H]5-HT, [ 3H]dopamine, [ 3H]noradrenaline and [ 3H]octopamine into the suboesophageal ganglia of the snail, Helix pomatia, was studied. When tissues were incubated at 25° in mediums containing radioactive amines, tissue: medium ratios of about 30:1, 18: l, 4:1 and 5:1 for 5-HT, dopamine, noradrenaline and octopamine respectively were obtained after 20–30 min incubation. Tissues incubated at 25° in mediums containing radioactive amines for 20–30 min showed that 90% of the radioactivity was present as unchanged [ 3H]5-HT, [ 3H]dopamine, [ 3H]noradrenaline or [ 3H]octopamine. The high tissue : medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, demonstrated saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver-Burk plots, two uptake mechanisms for 5-HT at 25° were resolved, the high affinity uptake process having a K m1 value of 8.48 × 10 −8 M and V max1 value of 0.077 nmole/g per min, while the lower affinity process had a K m 2 value of 1.8 × 10 −6 M and a V max2 value of 0.66 mole/g per min. At 0° a single uptake mechanism for 5-HT occurred which gave a K m value of 0.152 × 10 −8 M and a V m value of 0.0203 nmole/g per min. In the case of dopamine, the Lineweaver-Burk plot of 25° showed a single uptake process with values for K m and V max of 1.02 × 10 −7 M and 0.0673 nmole/g per min respectively. This process did not function at 0°. The effects of various agents and ions upon the accumulation processes for all amines were also studied, and the findings indicate that the same neurons may well accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the snail ganglia is a mechanism for inactivating these substances at 25° and that an uptake mechanism for 5-HT also functions at 0°. The present results are discussed from the point of view of the monoamines having transmitter functions in the snail CNS.

Glutamate uptake by chick retina.

The uptake of glutamate was found to be via a single high-affinity transport mechanism with Km values of 35 and 95 mum for chick-embryo and mature chick retina respectively. These data contrast with the uptake of gamma-aminobutyratewhich in the same tissue has previously been shown to display two kinetically distinct mechanisms in the embryo, but a single low-affinity process in the mature retina.

Binding and Degradation of Low Density Lipoproteins by Cultured Human Fibroblasts: COMPARISON OF CELLS FROM A NORMAL SUBJECT AND FROM A PATIENT WITH HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA

125I-Labeled low density lipoproteins were found to associate with monolayers of cultured normal fibroblasts by two processes—one of high affinity and one of low affinity. The high affinity association appeared to represent binding of the low density lipoprotein to specific receptor sites on the cell surface. This binding process exhibited saturation kinetics at low concentrations of the lipoprotein and competition by related molecules such as very low density lipoproteins. In addition, this process was stimulated by the presence of calcium in the culture medium and could be destroyed by limited treatment of the cells with pronase. The other process, designated low affinity uptake, may represent nonspecific endocytosis since the uptake was proportional to the lipoprotein concentration in the medium with no apparent saturation and because it showed no competition by very low density lipoproteins, no stimulation by calcium, and no destruction by pronase treatment. The 125I-labeled low density lipoproteins associated with normal cells by either the high or low affinity process were degraded by proteolysis to trichloroacetic acid-soluble material, most of which was 125I-tyrosine. In normal cells, binding of low density lipoproteins to the high affinity membrane receptor sites appears to serve two functions: (a) it results in suppression of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, and (b) it facilitates the degradation of low density lipoproteins when they are present in the culture medium at low concentrations (i.e. high affinity degradation). Cultured cells from subjects with the homozygous form of familial hypercholesterolemia, which were found to lack the high affinity binding process, were resistant to suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by low density lipoproteins and were also deficient in high affinity degradation. On the other hand, these mutant cells showed a normal low affinity uptake of low density lipoproteins and were able to degrade these lipoproteins when they were present in the culture medium at high concentrations. The possibility is raised, therefore, that a prerequisite for the regulation of cholestero-genesis in cultured fibroblasts is the initial binding of low density lipoproteins to the high affinity surface receptor sites and that a defect in this process represents the primary genetic abnormality in the disorder familial hypercholesterolemia.

Characteristics of tryptophan accumulation by glial cells

l-Tryptophan uptake was studied in C6 glial cells and found to be a very rapid and temperature dependent process. Kinetic analysis shows the presence of two saturable transport systems acting at different tryptophan concentrations. Both these high- and low-affinity processes were found in 3T3 fibroblast cultured cells, as well as in synaptosomes and cell suspensions obtained from rat cortex. No Na + requirement could be demonstrated in a wide range of tryptophan concentrations. The data presented here suggest that tryptophan accumulation in glial cells could be dependent on allosteric changes of the carrier, which might be relevant to the understanding of 5-HT synthesis regulation.

Kinetics of serotonin accumulation into synaptosomes of rat brain-effects of amphetamine and chloroamphetamines

Synaptosomes from whole brain of rats accumulated serotonin by means of a high affinity process and a low affinity process, distinguishable kinetically. The high affinity process was stronly inhibited by 4-chloroamphetamine. 3-Chloroamphetamine, 2-chloroamphetamine and amphetamine were progressively weaker inhibitors of that process. 4-Chloroamphetamine was a less potent inhibitor of the low affinity process (about equal to amphetamine). When 4-chloroamphetamine was administered to rats and synaptosomes were isolated from whole brain, the high affinity uptake of serotonin was inhibited for as long as 22·5 hr after drug administration. In vitro, 4-chloroamphetamine but not chloroimipramine at 5 μM caused a release of serotonin from synaptosomes preincubated with radioactive serotonin. The ability of 4-chloroamphetamine to inhibit the high affinity uptake of serotonin by synaptosomes and to release serotonin from synaptosomes is probably related to the prolonged lowering of brain serotonin levels in rats treated with 4-chloroamphetamine.