Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter‐like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high‐affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium‐3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na⁺‐independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H⁺ gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K⁺ concentration. In addition, the Na⁺‐independent uptake of choline was saturable, and the K_m value was estimated to be 108 µm . These findings suggest that the uptake of choline into LS180 cells is membrane potential‐dependent, but not outward H⁺ gradient‐dependent. Copyright © 2014 John Wiley & Sons, Ltd.     

Involvement of Recognition and Interaction of Carnitine Transporter in the Decrease of l-Carnitine Concentration Induced by Pivalic Acid and Valproic Acid

Purpose Prodrugs with pivalic acid and valproic acid decrease l-carnitine concentration in plasma and tissues by urinary excretion of acylcarnitine as pivaloylcarnitine (PC) and valproylcarnitine (VC), respectively. We investigated the role of the Na⁺/l-carnitine cotransporter in the porcine kidney epithelial cell line, LLC-PK₁ for the decrease of l-carnitine concentration.Methods The uptake of l-[³H]carnitine, acetyl-l-[³H]carnitine (AC), l-[³H]PC and l-[³H]VC were investigated in LLC-PK₁ cells seeded in a 6-well culture plate.Results l-Carnitine and AC uptake in LLC-PK₁ cells exhibited Na⁺ dependency. The K _m values for l-carnitine and AC uptake were 11.0 and 8.18 μM, respectively. These results indicated expression of Na⁺/l-carnitine cotransporter in LLC-PK₁ cells. PC and VC inhibited Na⁺/l-carnitine cotransporter in the competitive (K _i = 90.4 μM) and noncompetitive (K _i = 41.6 μM) manners, respectively. PC and VC uptake by Na⁺/l-carnitine cotransporter were not observed in LLC-PK₁ cells.Conclusions These data suggested that PC and VC formed in the body could not be reabsorbed in the kidney, resulting in the decrease of l-carnitine concentration. In addition, inhibition of l-carnitine reabsorption by VC with lower K _i value could induce the decrease of l-carnitine concentration. Collectively, the recognition and interaction of Na⁺/l-carnitine cotransporter are important factors for carnitine homeostasis.     

Inhibitory effects of noscapine on dopamine biosynthesis in PC12 cells

The effects of noscapine, a phthalide isoquinoline alkaloid, on dopamine biosynthesis and tyrosine hydroxylase (TH) activity in PC12 cells were investigated. Noscapine showed 74.6% inhibition on dopamine content in PC12 cells at a concentration of 20 μM. IC₅₀ of noscapine was 6.8 μM. TH activity was inhibited by the treatment of noscapine in PC12 cells (20.9% inhibition at 20 μM). Therefore, the inhibition of TH activity by noscapine might be involved in at least one component of the reduction of dopamine biosynthesis in PC12 cells.     

Synthesis and antitumor activities of unsymmetrically disubstituted acylthioureas fused with hydrophenanthrene structure

A series of novel unsymmetrically disubstituted acylthioureas fused with hydrophenanthrene structure were synthesized from Δ⁸-dihydroabietic and dehydroabietic acid, respectively. Their structures were characterized by IR, ¹H-, and ¹³C-NMR spectroscopy. The antitumor activities of the title compounds against SMMC7721 and A549 tumor cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results showed that three compounds (4c1, 4d1, and 4d2) exhibited highly effective activities against SMMC7721 and A549 cells, their IC₅₀ values are between 1.87–12.67 μM for SMMC7721 cells and 2.20–6.79 μM for A549 cells, respectively. Structure–activity relationship indicating that acylthioureas that furan group fused with Δ⁸-dihydroabietyl group, trihydroxymethyl fused with Δ⁸-dihydroabietyl and dehydroabietyl could generate enhance activities of this kind of compounds against SMMC7721 and A549 cells.     

C-type natriuretic peptide inhibits rat mesangial cell proliferation by a phosphorylation-dependent mechanism

We studied the effects of C-type natriuretic peptide (CNP) on rat cultured mesangial cell proliferation. (1) Exposure to CNP (10 nM–1 μM for 72 h) inhibited [³H]thymidine incorporation into mesangial cells in a concentration-dependent manner. Atrial natriuretic peptide (1 nM–1 μM), a peptide related to CNP, also decreased [³H]thymidine incorporation into these cells in a concentration-dependent manner. (2) Both CNP (10 nM-1 μM) and atrial natriuretic peptide (10 nM-1 μM) also decreased mesangial cell number. (3) The cyclic GMP analog, 8-bromo-cyclic GMP (100 μM and 1 mM), mimicked the inhibitory effects of CNP and atrial natriuretic peptide on [³H]thymidine incorporation into mesangial cells, whereas inhibitors of protein kinase C, protein kinase A, and protein kinase G reduced the effect of both natriuretic peptides. Moreover, the phoshpatase inhibitor, calyculin A, increased [³H]thymidine incorporation into mesangial cells. (4) CNP and atrial natriuretic peptide decreased interleukin-1-, interleukin-6-, platelet derived growth factor-, angiotensin II-induced [³H]thymidine incorporation into mesangial cells. These results suggest that CNP exerts inhibitory effects on mesangial cell proliferation and that this effects depend on protein phosphorylation pathways. Received: 17 March 1997 / Accepted: 29 August 1997     

Cannabinoid CB1 receptor-mediated inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain

Guinea-pig hippocampal slices preincubated with [³H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [³H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB₁ receptors was determined in rat brain cortex membranes using [³H]SR 141716. In guinea-pig hippocampal slices preincubated with [³H]noradrenaline, tritium overflow stimulated by NMDA 100 μM and 1000 μM and by kainate 1000 μM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB₁ receptor antagonist SR 141716 increased the NMDA (1000 μM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 μM or kainate 1000 μM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 μM)- and kainate (1000 μM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [³H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 μM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [ ³ H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pK _i 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pK _i < 5). In conclusion, activation of CB₁ receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB₁ receptors or that these receptors are activated by endogenous cannabinoids. Received: 25 February 1999 / Accepted: 12 April 1999     

α2-Adrenoceptors in opossum kidney cells couple to stimulation of mitogen-activated protein kinase independently of adenylyl cyclase inhibition

We have compared the effects of adrenaline on activation of mitogen-activated protein kinase (MAP kinase), cyclic AMP accumulation and [³H]thymidine uptake in OK cells, a cell line derived from proximal tubules of the opossum kidney. Effects of serotonin and the direct protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), were also studied. Adrenaline transiently (peak at 5min, return to baseline by 30min) and concentration-dependently (EC₅₀ between 10 and 100nM) stimulated MAP kinase activity. Maximal stimulation was approximately 100% above basal and was similar to the effects of 1μM serotonin or 1μM PMA. MAP kinase activation by adrenaline was inhibited by 10μM phentolamine or 1μM yohimbine but not significantly affected by 100nM prazosin or 200nM pindolol. The selective α₂-adrenoceptor agonist UK 14,304 (10μM) also stimulated MAP kinase activity. Activation of the 42 and 44kDa ERK forms of MAP kinase was demonstrated by immunoblot analysis. The effect of adrenaline and UK 14,304 on MAP kinase was inhibited by pertussis toxin pretreatment and by the MAP kinase kinase inhibitor, PD 98059 (100μM). Stimulation of MAP kinase activity was independent of cellular cAMP levels and was not affected by protein kinase C down-regulation. Adrenaline, UK 14,304, serotonin, and PMA stimulated [³H]thymidine uptake, an effect inhibited by PD 98059. We conclude that adrenaline stimulates MAP kinase activity in OK-cells via α₂-adrenoceptors and pertussis sensitive G proteins. While this occurs independently of cellular cAMP levels and protein kinase C, it involves the MEK1 form of MAP kinase kinase and the ERK forms of MAP kinase. This activation results in enhanced cellular proliferation as assessed by [³H]thymidine uptake. Received: 2 April 1997 / Accepted: 29 April 1997     

Mammalian glial cells in culture synthesize acetylcholine

In the present study we demonstrate that acetylcholine is synthesized by cultured mammalian glial cells identified by cell-type specific markers. Primary cultures of rat brain astrocytes or microglia contained 2.0 and 1.6 pmol acetylcholine/10⁶ cells on average respectively. Astrocyte cultures established from neonatal mouse brain contained even more acetylcholine (about 80 pmol acetylcholine/10⁶ cells). Primary cultures of rat brain astrocytes showed choline acetyltransferase (ChAT) enzyme activity of 3 nmol/mg protein/h; ChAT activity was blocked by 10 μM bromoacetylcholine. In conclusion, these data demonstrate the synthesis of the “neurotransmitter” acetylcholine in cultured glial cells, a finding which opens a new view upon the role of acetylcholine in mammalian brain.     

Effects of Various Methoxyflavones on Vincristine Uptake and Multidrug Resistance to Vincristine in <Emphasis Type="BoldItalic">P</Emphasis>-gp-Overexpressing K562/ADM Cells

Purpose Some methoxyflavones (MFs) are known to inhibit the function of P-glycoprotein. The aim of this study is to characterize the reversal of multidrug resistance (MDR) by MFs. Methods The effects of 19 MFs, including 3,5,6,7,8,3′,4′-heptamethoxyflavone, nobiletin, and tangeretin, and flavone on the uptake of [³H]vincristine into an adriamycin-resistant variant of human chronic myelogenous leukemia (K562/ADM) cells were investigated. Potentiation of vincristine-induced growth inhibition by these MFs was also tested in K562/ADM cells by means of WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] assay. Results All MFs (20 μM) tested increased the uptake of [³H]vincristine. 3,5,6,7,8,3′,4′-heptamethoxyflavone, nobiletin, tangeretin, quercetagetin and quercetin pentamethylether showed especially potent effects. The increase in the uptake of [³H]vincristine was proportional to the number of methoxyl moieties. While substitution with a methoxyl moiety at the C3 position was the most influential, methoxyl substitution at both the C3′ and C5′ positions resulted in a decrease in the potentiation of uptake. Furthermore, there was a significant correlation between the potencies for increasing [³H]vincristine uptake and for growth inhibition assessed by WST-1 assay. Conclusions MFs increased the uptake of [³H]vincristine into MDR cells and exhibited MDR-reversing effects. Their potencies were influenced by the number and positions of the methoxyl moieties.     

Cyclosporin A induces apoptosis in rat hepatocytes in culture

Cyclosporin A (CsA) at concentrations up to 1 μM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180–200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 μM CsA and diminished to 49% of control in the presence of 1 μM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-³H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 μM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes. Received: 17 February 1998 / Accepted: 20 April 1998     

Aniracetam, 1-BCP and cyclothiazide differentially modulate the function of NMDA and AMPA receptors mediating enhancement of noradrenaline release in rat hippocampal slices

Aniracetam, 1-(1,3-benzodioxol-5-yl-carbo-nyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the ‘kynurenate test’, a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-d-aspartate (NMDA)-evoked [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 μM) antagonism of the [³H]NA release elicited by 100 μM NMDA with high potency (EC₅₀≤0.1 μM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) added at 5 μM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of cyclothiazide. The AMPA-evoked release of [³H]NA from hippocampal slices or synaptosomes was enhanced by cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [³H]NA release in slices; this antagonism was attenuated by 1 μM cyclo-thiazide and reversed to an enhancement of AMPA-evoked [³H]NA release by 10 μM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP. Received: 14 September 1998 / Accepted: 19 January 1999     

Expression and Functional Characterization of Choline Transporter in Human Keratinocytes

Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na⁺, saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 μM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K⁺. These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.     

Modulation of aflatoxin B1 biotransformation by β-naphthoflavone in isolated rabbit lung cells

 The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B₁ (AFB₁) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB₁–8,9-epoxide. In a number of tissues and species, AFB₁ hydroxylation to the relatively nontoxic metabolite, AFM₁, is induced by β-naphthoflavone (BNF) treatment. Although the liver is the principal target organ for AFB₁ toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB₁ bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB₁ metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [³H]AFB₁. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45–50%) fractions, [³H]AFM₁ production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 μM [³H]AFB₁; control, 10.6±2.3; BNF, 30.0±6.4; with 0.10 μM [³H]AFB₁; control, 9.4±4.7; BNF, 20.6±5.9. With 1.0 μM [³H]AFB₁, prior treatment of animals with BNF abolished formation of [³H]aflatoxicol (AFL) but not [³H]AFQ₁. The activation (epoxidation) of [³H]AFB₁ was measured indirectly as covalent binding to endogenous DNA. With 1.0 μM [³H]AFB₁, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103±41; BNF, 114±49. However, with 0.10 μM [³H]AFB₁, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4±4.2; BNF, 9.3±3.9. Formation of 8,9-dihydro-8,9-dihydroxy- AFB₁, and the glutathione conjugate of the aflatoxin epoxide (AFB₁-GSH) were not detectable at the AFB₁ concentrations and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 μM BNF decreased [³H]AFB₁-DNA binding in the presence of 0.1 μM [³H]AFB₁ by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e.<0.5 μM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB₁-DNA adduct formation. The decrease in AFB₁-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM₁ formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB₁-8,9-epoxide with glutathione. Received: 5 March 1996/Accepted: 10 May 1996     

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion–cytochrome c-caspase protease mechanism.     

Efflux studies allow further characterisation of the noradrenaline and 5hydroxytryptamine transporters in rat lungs

The aim of the present study was to further characterise the noradrenaline and 5hydroxytryptamine [5HT] transporters in rat lungs by examining the efflux of noradrenaline and 5HT, respectively. Lungs from rats were isolated and perfused via the pulmonary artery. After loading the tissue with ³H5HT or ³Hnoradrenaline the efflux of the relevant amine from the lungs was examined for 1525min. The rate constant for efflux of ³H5HT increased by 81% when Na⁺ ions were removed from the perfusion solution; increased gradually when a selective 5HT transporter inhibitor, 200nM citalopram, was added to the perfusion solution for the final 6min of efflux; and increased markedly and rapidly when substrates of the 5HT transporter, tryptamine (18μM) and 7methyltryptamine (12μM), were added for the final 6min of efflux. These effects of the substrates were abolished by 1μM citalopram, but were not significantly affected by 1μM desipramine, a selective uptake₁ inhibitor. On the other hand, the previously described substrateinduced increase in the rate of efflux of noradrenaline was significantly reduced by desipramine but was unaffected by citalopram. The results show that efflux of 5HT is mediated only by the 5HT transporter, with no significant contribution of uptake₁, and efflux of noradrenaline from rat lungs is mediated only by uptake₁ and not by the 5HT transporter. The effects of dopamine on the efflux of noradrenaline over a concentration range of 100-600nM were investigated and the results showed that 50% of the maximal increase in the rate of efflux occurred at a concentration of 275nM. This value did not differ from the K_m for uptake of dopamine. This result implies that the only factor affecting the substrate-induced increase in noradrenaline efflux is the affinity of the substrate for uptake₁. The efflux of noradrenaline was also examined in the absence and presence of two concentrations of desipramine (0.35and 1.5μM). Analysis of these results showed that uptake₁ contributed approximately 81% and diffusion 19% to the total efflux of noradrenaline and that 90% of the total noradrenaline efflux was subject to reuptake by uptake₁ into the pulmonary endothelial cells. Received: 23 January 1997 / Accepted: 26 March 1997     

Blockade of voltage-sensitive sodium channels by NS-7, a novel neuroprotective compound, in the rat brain

The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [³H]batrachotoxinin A 20α-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 μM , while the compound was less effective in the cardiac myocytes (Ki = 13 μM). Aconitine, on the other hand, inhibited [³H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast, NS-7 had no affinity for [³H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 μM)-evoked glutamate release in a concentration-dependent manner, the IC₅₀ value of which was 7.7 μM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7. Received: 29 October 1996 / Accepted: 20 January 1997     

Membrane Binding Proteins are the Major Determinants for the Hepatocellular Transmembrane Flux of Long-Chain Fatty Acids Bound to Albumin

Purpose The hepatic transmembrane flux of long-chain fatty acids (LCFA) occurs through passive and fatty acid transport protein facilitated processes from blood. The extent that these transport processes can be related to the unbound and protein-bound fractions of LCFA in blood is not clear. Methods We used hepatocyte suspensions, hepatoma monolayers, and perfused rat livers to quantitate the transport of purified [³H]palmitate ([³H]PA) and 12-(N-methyl)-N-[(7-nitrobenz-2oxa-1,3-diazol-4yl-)amino]octadecanoicacid (12-NBDS) from solutions with a constant unbound LCFA concentration with varying bovine serum albumin (BSA) concentrations and in the presence and absence of antisera raised against cytosolic liver fatty acid binding protein (L-FABP). Results In the absence of L-FABP antisera, using an unbound ligand concentration that was adjusted to remain constant at each BSA concentration, hepatocyte [³H]PA and 12-NBDS uptake rates increased linearly with an increase in BSA concentration (p < 0.0001). In the presence of L-FABP antisera, [³H]PA uptake showed a greater reduction in the presence of 100 μM BSA than 5 μM BSA. The calculated permeability surface area product (PS) confirmed that both unbound and bound fractions of LCFA contributed to the overall flux, but only the PS for the protein-bound fraction was reduced in the presence of L-FABP antisera. In situ rat liver perfusion studies showed that the only rate process for the disposition of [³H]PA in the liver inhibited by L-FABP antisera was that for influx, as defined by PS, and that it reduced PS in the perfused liver by 42%. Conclusion These results suggest that, at physiological albumin concentrations, most of the LCFA uptake is mediated from that bound to albumin by a hepatocyte basolateral membrane transport protein, and uptake of unbound LCFA occurring by passive diffusion contributes a minor component.     

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine

The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.     

Contribution of equilibrative nucleoside transporter (ENT) 2 to fluorouracil transport in rat placental trophoblast cells

Fluorouracil is used for treatment of breast cancer even in pregnant women, except during fetal organogenesis. The purpose of this study was to clarify the transport mechanism of fluorouracil at the rat placental barrier. Maternal-to-fetal transfer of [³H]fluorouracil in rats at gestational day 19.5 was saturable and much higher than that of [¹⁴C]sucrose. The uptake of [³H]fluorouracil was also saturable in rat placental trophoblast TR-TBT 18d-1 cells, which express both equilibrative nucleoside transporter (ENT) 1 and ENT2. Nitrobenzylthioinosine (NBMPR) at 0.1 μM had no effect on [³H]fluorouracil uptake by TR-TBT 18d-1 cells, but 100 μM NBMPR almost completely inhibited the saturable component, suggesting involvement of ENT2, rather than ENT1 in the transport. Rat ENT2 cRNA-injected oocytes showed significantly increased [³H]fluorouracil uptake compared with water-injected oocytes, while rat ENT1 cRNA-injected oocytes did not show an increase of [³H]fluorouracil uptake. The Michaelis–Menten constant for rat ENT2-mediated uptake of [³H]fluorouracil was 4.21 mM. The expression profile of ENT2 mRNA in rat placenta during pregnancy was almost constant from 13.5 to 21.5 days of gestation. In conclusion, ENT2 appears to be the mediator of fluorouracil transport in rat placental trophoblast cells.     

Increased uptake of [3H]choline by rat superior cervical ganglion: An effect of dexamethasone

The high affinity uptake of [³H]choline by the superior cervical ganglion, isolated from the rat, was found to be increased by dexamethasone. Maximal increase (60–65% above control values) occurred at the steroid concentration of 5 × 10^?5 M. Other glucocorticoids (triamcinolone, corticosterone and hydrocortisone) were without an effect on the [³H]choline uptake. Following administration of dexamethasone (25 mg/kg, i.p.), there was a marked increase in the level of choline in the ganglion. The increase was 3-fold at 1 hr and 10-fold at 6 hr, and by 24 hr the choline levels still remained higher in the steroid-treated animals than in the controls. Levels of acetylcholine in the ganglion were also increased, beginning at thr after the injection of steroid. The increase was 85% by 3 hr and 60% by 6hr. Triamcinolone, a glucocorticoid that was without an effect on [³H]choline uptake in vitro, was also ineffective in altering the levels of choline and acetylcholine in vivo. It seems probable that the increase of choline uptake in the ganglion induced by dexamethasone may, at least in part, occur in the preganglionic cholinergic terminals, leading to increased synthesis of acetylcholine. Such an effect of dexamethasone provides another case of a selective steroid acting directly on nerve terminals by altering a transport mechanism.