Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.     

Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Acyl-based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations

Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl-based uptake inhibitors [N-(4-hydroxyphenyl)arachidonylamide (AM404), N-(4-hydroxy-2-methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) and (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-octadecen-amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 microm) and VDM11 (10 microm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5,000 per well were used. In contrast, UCM707 (30 microm), OMDM2 (10 microm) and the related compound arvanil (10 microm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6-9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl-based compounds was related primarily to the acyl side-chain rather than the head group. A variety of pre-treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N-acetyl-L-cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl-derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.     

Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells

The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear. We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells. CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance. SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM. CsA and SDZ 33-243 at 10 microM increased [3H]vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively. At 10 microM, these compounds also increased [3H]VCR accumulation by 3.5- and 4.0-fold, respectively. [3H]VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively. Kinetic analysis of [3H]VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity [3H]VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM. In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM. These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.     

Transport of organic cations by a renal epithelial cell line (OK)

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.     

Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

Anandamide uptake by human endothelial cells and its regulation by nitric oxide

Anandamide (AEA) has vasodilator activity, which can be terminated by cellular re-uptake and degradation. Here we investigated the presence and regulation of the AEA transporter in human umbelical vein endothelial cells (HUVECs). HUVECs take up AEA by facilitated transport (apparent K(m) = 190 +/- 10 nm and V(max) = 45 +/- 3 pmol. min(-1).mg(-1) protein), which is inhibited by alpha-linolenoyl-vanillyl-amide and N-(4-hydroxyphenyl)-arachidonoylamide, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scavenger hydroxocobalamin abolishes the latter effect, which is instead enhanced by superoxide anions but inhibited by superoxide dismutase and N-acetylcysteine, a precursor of glutathione synthesis. Peroxynitrite (ONOO(-)) causes a 4-fold activation of AEA transport into cells. The HUVEC AEA transporter contributes to the termination of a typical type 1 cannabinoid receptor (CB(1)) -mediated action of AEA, i.e. the inhibition of forskolin-stimulated adenylyl cyclase, because NO/ONOO(-) donors and alpha-linolenoyl-vanillyl-amide/N-(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, respectively, this effect of AEA. Consistently, activation of CB(1) cannabinoid receptors by either AEA or the cannabinoid HU-210 caused a stimulation of HUVEC inducible NO synthase activity and expression up to 2.9- and 2. 6-fold, respectively. Also these effects are regulated by the AEA transporter. HU-210 enhanced AEA uptake by HUVECs in a fashion sensitive to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester. These findings suggest a NO-mediated regulatory loop between CB(1) cannabinoid receptors and AEA transporter.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene.

Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction.     

Pharmacological Characterization of Rapidly Accumulated Adenosine by Dissociated Brain Cells from Adult Rat

Mechanically dissociated brain cells from adult rats were used to study biochemically and pharmacologically their capacity to accumulate rapidly [3H]adenosine. The assay, which used an inhibitor-stop method to prevent further uptake into cells, was characterized with respect to protein and optimal substrate concentrations, and incubation times that ranged from 5 to 180 s. The accumulation of [3H]adenosine using 15-s incubation periods, conditions under which less than 10% of accumulated [3H]adenosine was metabolized, was best described kinetically by a two-component system with Km and Vmax values for the high-affinity component of 0.8 microM and 6.2 pmol/mg protein/15 s and for the low-affinity component 259 microM and 2,217 pmol/mg protein/15 s, respectively. The potencies with which nucleosides, adenosine deaminase resistant adenosine receptor agonists, and nucleoside uptake inhibitors competed for these uptake components were determined. Of the nucleosides examined, adenosine was the "preferred" substrate for the uptake site. The Ki value of adenosine for the high-affinity component was 10.7 microM. Inosine and uridine competed for a single lower affinity uptake system: Ki values were 142 and 696 microM, respectively. Nucleoside uptake inhibitors--nitrobenzylthioinosine, dipyridamole, and dilazep--were the most potent inhibitors of [3H]adenosine accumulation tested: the Ki values for the high-affinity system were 0.11, 1.3, and 570 nM, respectively. The adenosine analogs S-phenylisopropyladenosine, R-phenylisopropyladenosine, and cyclohexyladenosine inhibited the high-affinity component with Ki values of 2.3, 9.3, and 14.5 microM, respectively. N-Ethylcarboxamidoadenosine competed for a single lower affinity uptake system: Ki, 292 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choline Uptake by Cerebral Capillary Endothelial Cells in Culture

A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood-brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier-mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a Km of 7.59 +/- 0.8 microM and a maximum capacity of 142.7 +/- 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1-100 microM). When cells were preincubated in the presence of choline, a single saturable component was observed with a Km of 18.5 +/- 0.6 microM and a maximum capacity of 452.4 +/- 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium-3, deanol, and AF64A. The presence of ouabain or 2,4-dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine-5'-diphosphocholine and phospholipids; however, under steady-state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

Carrier-Mediated Transport of Thyroid Hormones into Rat Glial Cells in Primary Culture

The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited.     

Inhibition by Anandamide and Synthetic Cannabimimetics of the Release of [3H]d-Aspartate and [3H]GABA from Synaptosomes Isolated from the Rat Hippocampus

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K(+)-evoked release of [3H]D-aspartate ([3H]D-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]D-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2'-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]D-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.     

Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus

This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high- and a low-affinity uptake mechanism. The high-affinity mechanism had a Km of 25.2-30.5 microM and a Vmax of 3.8-4.8 nmol/10 mg of cell water/5 min, whereas the low-affinity mechanism had a Km of 633-718 microM and a Vmax of 26.6-37.1 nmol/10 mg of cell water/5 min. At steady state, the high-affinity mechanism accumulated 10 microM [3H]glycine from the medium, achieving tissue concentrations that were 13-24 times that in the medium. The high-affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+-dependent release and the high-affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision.