Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

The inhibition of PB125I formation in calf thyroid caused by 14-iodo-15-hydroxy-eicosatrienoic acid is due to decreased H2O2 availability

Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-HO-A) is a potent inhibitor of iodine organification in calf thyroid slices. The present studies were performed in order to clarify the mechanism of this action. Incubation of thyroid slices with 10(-4)M I-HO-A caused a 47 and 53% decrease in PB125I formation after 30 and 60 min incubation, respectively. In a series of experiments an inverse relationship between the degree of inhibition caused by I-HO-A and total iodine content and basal iodoprotein formation was observed. Chromatographic analysis of the labeled compounds showed a significant decrease in 125I incorporation into MIT, DIT, T3 and total iodolipid. The site of the inhibitory effect of I-HO-A was then sought. TPO was measured by three different methods. When TPO was solubilized from I-HO-A treated slices, no change in enzymatic activity was observed. Moreover, the same lack of action was found when solubilized TPO was incubated with I-HO-A. The production and release of H2O2 into the incubation medium was measured by chemiluminiscence technique. In control slices the values increased during the first 10 min and reached a plateau. Pretreatment of the slices with 10(-4)M KI caused a 51% inhibition, while the same concentration of I-HO-A produced a 59% inhibition. The possibility that I-HO-A might exert its action through a putative protein inhibitor was also explored. Incubation of slices with 10(-5)M I-HO-A caused a 46% decrease in PB125I formation and addition of actinomycin D or puromycin failed to alter this effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.     

Identification of a major iodolipid from the horse thyroid gland as 2-iodohexadecanal

The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of active iodide transport by NaCIO4 reduced both PBI and LBI. Chromatography on silica gel resolved almost equal amounts of low and high polarity iodolipids. The main unpolar iodolipid was identified as 2-iodohexadecanal (2-IHDA), on the basis of proton nuclear magnetic resonance spectroscopy, mass spectrometry, and co-elution with authentic 2-IHDA obtained by chemical synthesis in reversed-phase high performance liquid chromatography and gas chromatography. The presence of 2-IHDA was also detected in dog thyroid slices, following incubation with KI (50 microM) and in the rat thyroid, 4 hours after intraperitoneal injection of KI (650 micrograms). An incubation of bovine brain plasmalogens with lactoperoxidase, iodide, and H2O2 generated 2-IHDA. In conclusion, we have identified a major thyroid iodolipid as 2-iodohexadecanal. The biosynthesis of this compound is likely to involve the addition of iodine to the vinyl ether group of plasmalogens.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-omega); 3) 5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-delta). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10(-4)M I-OH-A caused a 64% decrease in the T/M ratio, while IL-omega inhibited it by 36% and IL-delta was without effect. At 10(-5)M the inhibition was 44% for I-OH-A and 19% for IL-omega, while T3 was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodide uptake was observed after only 15 min preincubation. This compound also decreased 125I accumulation in rats. In calf thyroid slices, I-OH-A at 10(-4)M, inhibited PB125I formation by 80%, IL-omega by 62% and IL-delta by 37%. T3 and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification. The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibit thyroid function and mimic the effect of iodide on thyroid autoregulation.     

The effect of long-term external ionizing radiation on the functional activity of rat thyroid under enhanced potassium iodide consumption

The study was devoted to the effect of long-term (20 days) external ionizing radiation at a dose of 0.5 Gy on the iodide metabolism in the rat thyroid under supplementation of high iodine doses (10 daily KI doses). It was found that the potassium iodide administration partially prevented the effects of a post radiation decrease of serum thyroid hormone levels (the level of T4 was normal and that of T3 was 77.4% of the controls). After the supplementation of 10 daily iodide doses, the rat thyroid tissue showed the most pronounced increase in the levels of total, free and protein-bound iodide compared to the groups of animals consuming normal and elevated KI doses. Pronounced inhibition of thyroid peroxidase activity (3.1-fold) was noted in the same group. The data obtained indicate a radiation-induced activation of iodide uptake during its enhanced supplementation and disturbed iodide enzymatic oxidation and organification.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

From the molecular characterization of iodide transporters to the prevention of radioactive iodide exposure

In the event of a nuclear reactor accident, the major public health risk will likely result from the release and dispersion of volatile radio-iodines. Upon body exposure and food ingestion, these radio-iodines are concentrated in the thyroid, resulting in substantial thyroidal irradiation and accordingly causing thyroid cancers. Stable potassium iodide (KI) effectively blocks thyroid iodine uptake and is thus used in iodide prophylaxis for reactor accidents. The efficiency of KI is directly related to the physiological inhibition of the thyroid function in the presence of high plasma iodide concentrations. This regulation is called the Wolff-Chaikoff effect. However, to be fully effective, KI should be administered shortly before or immediately after radioiodine exposure. If KI is provided only several hours after exposure, it will elicit the opposite effect e.g. lead to an increase in the thyroid irradiation dose. To date, clear evaluation of the benefit and the potential toxicity of KI administration remain difficult, and additional data are needed. We outline in this review the molecular characterization of KI-induced regulation of the thyroid function. Significant advances in the knowledge of the iodide transport mechanisms and thyroid physiology have been made. Recently developed molecular tools should help clarify iodide metabolism and the Wolff-Chaikoff effect. The major goals are clarifying the factors which increase thyroid cancer risk after a reactor accident and improving the KI administration protocol. These will ultimately lead to the development of novel strategies to decrease thyroid irradiation after radio-iodine exposure.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Propranolol has direct antithyroid activity: Inhibition of iodide transport in cultured thyroid follicles

The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier-free 0·1 μCi Na ¹²⁵I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose-dependent manner; this inhibition was non-competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta-blocking activity, since D -propranolol (devoid of beta-blocking activity) inhibited iodide transport, and other beta-blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH-mediated cAMP generation and Na ⁺K⁺ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol. Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Preservation of sodium-dependent iodide transport activity by methimazole and mercaptoethanol in phospholipid vesicles containing thyroid plasma membranes: with evidence of difference in the action of perchlorate and thiocyanate

The effect of methimazole (MMI) and 2-mercaptoethanol (ME) on I-transport was studied using phospholipid vesicles (P-vesicles) made from porcine thyroid plasma membranes and soybean phospholipids by sonication. 1. When buffer solutions contained either 1 mM MMI or 2 mM ME, I-uptake by P-vesicles in the presence of external Na+ was apparently higher than that in the absence of external Na+. Na+-dependent I- uptake was inhibited by both C1O4- and SCN- added externally. 2. When PM was treated with 4 mM N-ethylmaleimide prior to preparation of P-vesicles, the activity of Na+-dependent I- transport was completely lost even when P-vesicles were incubated in the presence of ME. 3. When neither MMI nor ME was added to buffers, I- uptake in the presence of external Na+ was not at all higher than that in the absence of external Na+. In these instances, however, I- uptake was much higher compared than the baseline uptake in the presence of MMI or ME, and was inhibited by external SCN- and not by C1O4- without relation to external Na+. These data indicate that MMI or ME has two distinct effects on our model system of I- transport. The one is preservation of the Na+-dependent I- transport activity by protecting a sulfhydryl group, and the other is reduction of nonspecific I- binding to P-vesicles. In addition, C1O4- is a more specific inhibitor of thyroid I- transport than SCN-, when non-specific I- oxidation is imperfectly prevented.     

Inhibition of alpha-aminoisobutyric acid uptake by mastoparan in Madin-Darby Canine Kidney cells

Mastoparan, a wasp venom, was found to inhibit Na(+)-dependent net alpha-aminoisobutyric acid (AIB) uptake in Madin Darby Canine Kidney (MDCK) cells. Mastoparan also produced a significant increase in AIB efflux when compared to controls. Pretreatment of MDCK cells with 2 mM neomycin attenuated mastoparan's inhibition of net AIB uptake and completely suppressed mastoparan-mediated increases in AIB efflux when compared to controls. These data suggest that mastoparan's inhibition of net AIB uptake involves more than a single basic mechanism.     

2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers

2-Aminooxyisobutyric acid (AOIB) has a partial structure of aminooxyacetic acid (AOA) in its whole structure, and resembles 2-aminoisobutyric acid (AIB) in their tetrahedral structures. Both AOA and AIB are inhibitors of ethylene biosynthesis; AOA inhibits the action of 1-aminocyclopropane-1-carboxylate (ACC) synthase and AIB inhibits that of ACC oxidase. The present study showed that AOIB inhibited the in vitro activities of both ACC synthase and ACC oxidase, which were synthesized heterologously in E. coli cells from corresponding carnation cDNAs, and the magnitudes of inhibition were similar to those caused by AOA and AIB; AOIB and AOA at 0.1 mM inhibited ACC synthase action by 75%, and AOIB and AIB at 10 mM inhibited ACC oxidase action by 16.3 and 22.5%, respectively. AOIB at 1 mM caused 91.5% reduction of maximum ethylene production rate as compared to the control in cut ‘Excerea’ carnation flowers undergoing senescence, thereby lengthening their vase life to 7 d from 3 d of the control flowers. The inhibition by AOIB was probably caused by its action resembling AOA, but not AIB. AOIB also extended significantly the vase life of cut flowers of ‘Pax’ carnation, and tended to do so in ‘Primero Mango’ carnation. The present findings suggest the potential of AOIB as a new preservative for carnations and other ornamentals in which ethylene plays a key role in the induction of senescence.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Induction of amino acid transport by L-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)

The action of L-triiodothyronine (T3) on amino acid transport in the GC clonal strain of rat pituitary cells was investigated by measurement of the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). The uptake of AIB by GC cells appeared to require energy and Na+ and displayed Michaelis-Menten kinetics. In comparison to cultures maintained in the absence of T3, T3 addition resulted in an increase in AIB uptake which seemed due to an increase in the initial rate of AIB transport. T3 addition resulted in increased AIB accumulation at later time points as well. T3 induction of AIB transport did not occur until 3.5 h after addition of T3, and this effect was blocked by cycloheximide. Maximal induction occurred 48 to 72 h later. One-half maximal induction occurred 24 to 48 h after addition of T3. No detectable changes either in AIB uptake or intracellular water space, measured by uptake of the nonmetabolizable sugar, 3-O-methyl-D-glucose, were noted for the first 120 min after addition of T3. Induction of AIB transport occurred at 0.05 nM T3 (total medium concentration) and one-half maximal induction occurred at 0.17 nM T3. The relative potencies of four iodothyronine analogues for AIB transport were in accord with their reported activities in nuclear T3 receptor binding assays. These data suggest that induction of AIB transport by T3 may be mediated by the nuclear T3 receptor and may reflect the pleiotrophic response of GC cells to thyroid hormone.     

System A transport activity in normal rat hepatocytes and transformed liver cells: substrate protection from inactivation by sulfhydryl-modifying reagents

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid-dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.     

Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro.

Mitotic spindle inhibitors (colchicine, vinblastine, vincristine, 020, ethanol) and cytochalasin B inhibit the phagocytosis of colloid by thyroid cells and the secretion of thyroid hormones. This inhibition has been linked to interferences with the microtubular microfilament system of the follicular cell. In order to test the possibility of using such inhibitors to selectively block secretion, the action of suppressing or highly inhibitory concentrations on other metabolic parameters has been studied on dog thyroid slices in vitro: glucose oxidation, lactate formation, iodide binding to protein, cyclic 3'5' AMP accumulation. It is shown that at a concentration of 10 mM colchicine is entirely non specific as it greatly inhibits all facets of metabolism and all the stimulatory effects of cyclic 3'5' AMP and thyrotropin. The other mictrotubule inhibitors, although affecting thyroid metabolism in various ways were more specified. The enhancement by vineblastine of glucose oxidation ald iodine binding to proteins suggests an activation of they thyroid H2O2 generating system. D2O on the other hand selectively inhibits secretion and the binding of iodide to proteins. Cytochalasin B, presumably by inhibiting hexose transport, decreased glycolysis and the uptake of iodide. However this effect cannot account for the complete inhibition of thyroid secretion.     

Functional and molecular characteristics of system L in human breast cancer cells

The functional and molecular properties of system L in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na(+)-free conditions. alpha-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by L-alanine (83.6%), L-lysine (75.6%) but not by L-proline. Similarly, L-lysine and L-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K(m) of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V(max) was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, L-glutamine, L-alanine, L-leucine, L-lysine and AIB (all at 2 mM). In contrast, L-glutamate, L-proline, L-arginine and MeAIB had no effect. The interaction between L-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by D-leucine and D-tryptophan but not by D-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na(+)-free conditions is predominantly via system L which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.