Sorafenib-Induced Changes in Thyroid Hormone Levels in Patients Treated for Hepatocellular Carcinoma.

Context: The pathogenesis of tyrosine kinase inhibitor-induced thyroid hormone (TH) alterations are still a matter of debate. Objective: The objective of this study was to determine the effects of sorafenib on TH levels in patients with hepatocellular carcinoma (HCC) and to evaluate possible mechanisms. Design: We performed a prospective cohort study between 2009 and 2016. Setting: This study was conducted at a tertiary referral center. Patients: This study included 57 consecutive patients with HCC who were treated with sorafenib. Main Outcome Measure: Thyroid-stimulating hormone (TSH) and free thyroxine (FT4) levels were measured every 6 weeks, and extensive thyroid function tests (TFTs) were measured before treatment (t0), after 6 weeks (t6), and at the end of therapy. The effect of sorafenib on TH transport by monocarboxylate transporter (MCT)8 or MCT10 was tested in transfected COS1 cells. Results: Four patients (7%) developed thyroiditis. Among the other patients, 30% had elevation of TSH or FT4 above the normal range. Overall, between t0 and t6, mean TSH increased from 1.28 to 1.57 mU/L (P < 0.001) and mean FT4 from 18.4 to 21.2 pmol/L (P < 0.001). Simultaneously, the serum triiodothyronine (T3)/reverse triiodothyronine ratio and the (T3/thyroxine) ×100 ratio decreased. Sorafenib decreased cellular T3 uptake by MCT8 and to a lesser extent by MCT10. Conclusions: These in vivo data suggest that sorafenib affects TFTs on multiple levels. Our in vitro experiments suggest a possible role of sorafenib-induced inhibition of T3 transport into the cell by MCT8 and MCT10.

Isolation and characterization of proline/betaine transporter gene from oil palm.

Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm; the mechanism for this is unknown. Here, an 8319-nucleotide sequence including cDNA, genomic DNA and the promoter region of proline transporter gene from oil palm Elaeis guineensis was determined. The transporter gene exhibited high similarity to Bet/ProT genes from several plants, but the highest homology was found with rice ProT1. The exon-intron structure of genomic DNA was unique, and numerous stress-response cis-elements were found in the promoter region. Expression of cDNA EgProT1 in Escherichia coli mutant exhibited uptake activities for glycinebetaine and choline as well as proline. Under salt-stressed conditions, exogenously applied glycinebetaine was taken up into the root more rapidly than the control. These data indicate that oil palm has a unique Pro/T1 gene. Nucleotide sequence data for the cDNA and genomic DNA of proline transporter gene from Elaeis guineensis are available in the DDJB database under accession numbers AB597035 and AB597036, respectively.

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.

A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).

Enhancement of the NMDA receptor function by reduction of glycine transporter-1 expression.

The occupation of the glycine binding-site is a prerequisite for NMDA receptor activation by glutamate. To analyze the regulation of NMDA receptor function by the glycine transporter 1 (GlyT1), we generated heterozygous constitutive GlyT1 knockout mice (GlyT1tm1.1(+/-)). These animals were fully viable. Using a newly generated antibody, the pattern of GlyT1 expression in brain was found to be unaltered in the mutants while the level of expression was strongly reduced in all brain regions, as shown immunohistochemically. In hippocampal slices the ratio of the peak amplitude of NMDA and AMPA receptor evoked excitatory postsynaptic currents (EPSCs), recorded in CA1 pyramidal cells, was significantly enhanced by 36% in Glyt1tm1.1(+/-) compared to wild-type slices. The frequency and amplitude of AMPA miniature events in Glyt1tm1.1(+/-) mice were indistinguishable from those recorded in wild type. These results provide proof that the NMDA receptor function is enhanced by a reduction of GlyT1 expression. Thus, GlyT1 function is a controlling factor for an enhancement of the NMDA receptor response. These findings are of relevance for the development of GlyT1 inhibitory drugs.

Irreversible inhibition of [3H]glycine transport into channel catfish erythrocytes by thiol group modifiers.

Erythrocytes can take up amino acids from the blood by using a variety of transport systems. GLYT is a key transport protein in the plasma membrane responsible for the Na(2+)-dependent uptake of glycine needed for glutathione biosynthesis. Certain cysteine-specific compounds, particularly mercuric chloride and 5,5'-dithiobis(2-nitrobenzoate), irreversibly inhibited the [(3)H]glycine transport via GLYT by red blood cells isolated from channel catfish. Bimolecular rate constants (k(2)) of 0.556 (mmol/l)(-1) min(-1) and 0.032 (mmol/l)(-1) min(-1), respectively, were calculated for the two inhibitors. Addition of 2-mercaptoethanol 1 min after the initiation of inactivation by mercuric chloride stopped further inactivation, but did not reverse the inhibition. The presence of glycine, but not Na(+) ions, during the preincubation of the cells with each inhibitor markedly reduced the degree of inhibition. Thus cysteinyl residues within the transport protein appear to be vital for the binding and uptake of glycine by channel catfish erythrocytes.

Identification of a Na+-dependent cationic and neutral amino acid transporter, B(0,+), in human and rabbit cornea.

The purpose of this study was to identify and functionally characterize an active transport system for L-arginine on rabbit corneal epithelium and human cornea and study its interaction with the amino acid ester prodrugs of acyclovir (Anand, B. S.; Mitra, A. K. Pharm. Res. 2002, 19, 1194-1202). Transport characteristics of [3H]-L-arginine across freshly excised rabbit corneas were determined at various concentrations, in the absence of sodium and chloride ions. Inhibition studies were conducted in the presence of other amino acids, ouabain, and amino acid ester prodrugs of acyclovir (glycine-ACV, phenylalanine-ACV and gamma-glutamate-ACV). Reverse transcription-polymerase chain reaction (RT-PCR) for amino acid transporter B(0,+) was carried out on total RNA isolated from rabbit cornea, rabbit corneal epithelium, and human cornea. Transport of L-Arg across rabbit cornea was saturable (Km = 306 +/- 72 microM and Vmax = 0.12 +/- 0.01 nmol min(-1) cm(-2)) and was Na+, Cl-, and energy dependent. Transport was inhibited by neutral and cationic amino acids and a B(0,+) system specific inhibitor, BCH (Sloan, J. L.; Mager, S. J. Biol. Chem. 1999, 274, 23740-23745), but not by anionic amino acids. Amino acid prodrugs of ACV (Glu-ACV and Phe-ACV) also inhibited transport of [3H]-L-Arg across rabbit cornea. Amino acid transporter B(0,+) was identified by RT-PCR and its identity confirmed by subcloning and sequencing in rabbit cornea, rabbit corneal epithelium, and human cornea. A Na+-, Cl(-)-, and energy-dependent carrier for L-Arg, B(0,+), was identified on rabbit corneal epithelium and human cornea. Glu-ACV and Phe-ACV appear to be substrates for this transporter. The presence of such transporters on the corneal epithelium may provide new opportunities for transporter-targeted prodrug design for enhanced corneal absorption.

Dietary protein reduction in sheep and goats: different effects on L-alanine and L-leucine transport across the brush-border membrane of jejunal enterocytes.

It was the aim of this study to examine the potential regulatory effects of a long-term low dietary protein supply on the transport capacity of the jejunal brush-border membrane for amino acids. For this purpose, we used the neutral amino acids L-alanine (representative for nonessential amino acids) and L-leucine (representative for essential amino acids) as model substances. Ten sheep lambs, 8 weeks of age and 19-27 kg body weight, were allotted to two dietary regimes with either adequate or reduced protein supply which was achieved by 17.9% and 9.7% of crude protein in the concentrated feed, respectively. The feeding periods were 4-6 weeks in length. Similarly, eight goat kids of 5-7 weeks of age and 8-14 kg body weight were allotted to either adequate (crude protein 20.1%, feeding period 9-12 weeks) or reduced protein supply (10.1%, feeding period 17-18 weeks). Dietary protein reduction in lambs caused a significant body weight loss of 0.6 +/- 0.7 kg, whereas the body weight in control animals increased by 1.9 +/- 0.7 kg (P<0.05). Plasma urea concentrations decreased significantly by 60% (low protein 2.3 +/- 0.1 versus control 5.7 +/- 0.2 mmol l(-1), P<0.001). In kids, reduction of dietary protein intake led to significant decreases of the daily weight gain by 48% from 181 +/- 8 g to 94 +/- 3 g (P<0.001) and daily dry matter intake by 27% from 568 +/- 13 g to 417 +/- 6 g (P<0.01). Respective urea concentrations in plasma were reduced by 77% from 5.2 +/- 0.4 to 1.2 +/- 0.2 mmol l(-1) (P<0.01). Kinetic analyses of the initial rates of alanine uptake into isolated jejunal brush-border membrane vesicles from sheep and goats as affected by low dietary protein supply yielded that the apparent Km was neither significantly different between the species nor significantly affected by the feeding regime thus ranging between 0.12 and 0.16 mmol.l(-1). Reduction of dietary protein, however, resulted in significantly decreased Vmax values of the transport system by 25-30%, irrespective of the species. Kinetic analyses of the initial rates of leucine uptake into jejunal brush-border membrane vesicles from sheep and goats yielded that leucine uptake was mediated by Na+-dependent as well as Na+-independent processes. Similar to alanine, apparent Km values of leucine uptake were neither different between the species nor affected due to low dietary protein and ranged between 0.08 and 0.15 mmol l(-1). In contrast to the alanine transport mechanism, dietary protein reduction resulted in increased Vmax values of Na+-dependent leucine transport by 53% in sheep and 230% in goats. Similarly, Na+-independent leucine uptake was stimulated by 85% and 200% in sheep and in goats, respectively. This study shows adaptation of amino acid absorption at the brush-border membrane level of jejunal enterocytes of small ruminants due to dietary protein reduction. Whereas the transport capacity for the nonessential amino acid alanine was reduced due to low dietary protein, the transport capacity for the essential amino acid leucine was markedly stimulated. From this, the involvement of rather different feedback mechanisms in adaptation of intestinal amino acid transport mechanisms has to be discussed.

Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter, GLYT1a.

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.

Glycine receptors and transporters on bullfrog retinal Müller cells.

Glycine is a principal inhibitory neurotransmitter in the vertebrate retina. We have characterized glycine-induced currents from Müller cells in the bullfrog retinal slice preparation using whole-cell recordings. The glycine-induced current was partially suppressed by strychnine, and the remaining strychnine-resistant component was sacrosine-sensititve, suggesting that these two components may be mediated by glycine receptors and glycine transporters, respectively. Furthermore, the two components were maximal at the inner nuclear layer (INL) and declined asymmetrically as the application site was moved away from the INL to either the external or internal limiting membranes. It is suggested that Müller cells may be involved in glycinergic transmission by communicating with retinal neurons through these receptors and transporters.

Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels.

Using isolated bovine brain microvessels as an in vitro model of the blood-brain barrier (BBB) we have evaluated the role of free radical generating solutions on some amino acid transport systems operating on the endothelial cell membrane. Fe(2+)/ascorbate, phenylhydrazine and CuSO(4) did not affect any of the transport system tested, while exposure of bovine brain microvessels to tert-butylhydroperoxide (t-BHP) caused a reduced capacity to take up small neutral amino acids via the Na(+)-dependent A-system. The presence of glucose during t-BHP treatment did not prevent this inhibition, which was partially counteracted when the isolated microvessels were incubated with 5mM inosine before the oxidative stress. Incubation of the isolated capillaries with 5mM dithiothreitol, after exposure to t-BHP, resulted in a 50% recovery of the alpha-methylaminoisobutyrate (MeAIB) uptake by the A-system. Treatment with t-BHP, which had no effect on the L-system of neutral amino acid transport, caused a significant decrease of the intracellular levels of ATP, of glutathione (GSH), and of gamma-glutamyltranspeptidase (GGT) activity, while no significant modification of hexokinase (HK) or of alkaline phosphatase (ALKP) activities were observed. Oxidative damage of the BBB appears therefore to impair essentially the metabolic pathways which ensure the energy requirement for the endothelial cells, thus inhibiting the energy-dependent amino acid transport system "A".