Nicergoline enhances glutamate uptake via glutamate transporters in rat cortical synaptosomes

To elucidate the mechanisms of neuroprotective action of nicergoline, we examined its effect on glutamate transport in rat cortical synaptosomes and cloned glutamate transporters. In synaptosomes, nicergoline enhanced the glutamate uptake at 1-10 microM in standard medium and suppressed the increase of extracellular glutamate by reversed transport in low Na(+) medium. Apparent increase of extracellular glutamate concentration by dihydrokinate, an inhibitor of glial glutamate transporter GLT-1, was antagonized by nicergoline. In Xenopus oocytes expressing mouse neuronal glutamate transporter (mEAAC1), the glutamate-induced inward current was enhanced by nicergoline. These results suggest that nicergoline reduces the extracellular glutamate concentration through its effect on glutamate transporters.     

Release of L-aspartate by reversal of glutamate transporters

Aspartate is released in the brain during metabolic inhibition and can activate NMDA receptors. We compared the characteristics of aspartate and glutamate release mediated by reversed operation of GLAST glutamate transporters in salamander retinal glial cells, when high [K(+)](o) solution was applied to mimic the ionic conditions of stroke or glaucoma. In the absence of Cl(-), to isolate the transport-associated current of the transporters, reversed uptake of aspartate and glutamate had similar characteristics. Both were increased strongly by depolarisation, inhibited by the transport inhibitor TBOA (DL-threo-beta-benzyloxyaspartate), and activated in a first order manner by intracellular amino acid (in the presence of 20mM [Na(+)](i)) with an EC(50) of 0.8mM for aspartate and 2.3mM for glutamate. In stroke the extracellular pH shifts acid by around a pH unit: this reduced the release of aspartate and glutamate by reversed uptake by a factor of 8-20. The external Cl(-) concentration had only a small effect on the current associated with reversed uptake of aspartate and glutamate. Tamoxifen, which reduces amino acid release through swelling-activated anion channels in glial cells, was found to inhibit reversed uptake with an IC(50) which was >100 microM. Part of the activation of NMDA receptors which occurs in ischaemia is likely to reflect the release of aspartate by reversed uptake.     

Nicergoline enhances glutamate re-uptake and protects against brain damage in rat global brain ischemia

Whereas a 2-3 degrees C decrease in intraischemic brain temperature can be neuroprotective, mild brain hyperthermia significantly worsens outcome. Our previous study suggested that an ischemic injury mechanism which is sensitive to temperature may not actually increase the extracellular glutamate concentration ([Glu](e)) during the intraischemic period, but rather impairs the Glu re-uptake system, which has been suggested to be involved in the reversed uptake of Glu. We speculated that enhancing Glu re-uptake, pharmacologically or hypothermically, may shorten exposure to high [Glu](e) in the postischemic period and thereby decrease its deleterious excitotoxic effect on neuronal cells. In the present study, rats treated with nicergoline (32 mg/kg, i.p.), an ergot alkaloid derivative, showed minimal inhibition of the [Glu](e) elevation which characteristically occurs during the 10-min intraischemic period, while Glu re-uptake was dramatically improved in the postischemic period, when severe transient global ischemia was caused by mild hyperthermia. Moreover, the nicergoline (32 mg/kg, i.p.) treated rats showed reduced cell death morphologically and clearly had a far lower mortality. The present study suggests that the development of therapeutic strategies aimed at inhibition or prevention of the reversed uptake of glutamate release during ischemia, i.e., activation of the glutamate uptake mechanism, is a promising approach to reduce neural damage occurring in response to brain ischemia.     

Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, [3H]alanine transport was studied in plasma membrane vesicles. [3H]Alanine uptake was stimulated by an "out-to-in" Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-[3H]alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity. Inactivation of the Na(+)-coupled alanine carrier by mercury in hepatocytes of this evolutionarily primitive vertebrate, as in mammals, suggests that the sulfhydryl groups on this transport protein are highly conserved.     

Cerebral cystine uptake: a tale of two transporters

Transport of cystine across the cell membrane is essential for synthesis of the major cellular antioxidant glutathione. Cystine uptake in the brain occurs by both the Na(+)-independent x(c)(-) cystine-glutamate exchanger and the X(AG)(-) family of high-affinity, Na(+)-dependent glutamate transporters. New evidence concerning the role of cystine transport in the defence against oxidative stress is described.     

Effects of glutamate transport substrates and glutamate receptor ligands on the activity of Na-/K(+)-ATPase in brain tissue in vitro

1. It has been suggested that Na+/K(+)-ATPase and Na(+)-dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K(+)-ATPase activity using Rb+ uptake by 'minislices' (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy. 2. We determined the potencies of several known substrates/inhibitors of GluT, such as L-trans-pyrrolidine-2,4-dicarboxylate (LtPDC), DL-threo-3-benzyloxyaspartic acid, (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and L-anti,endo-3,4-methanopyrrolidine dicarboxylic acid, as inhibitors of [3H]-L-glutamate uptake by cortical prisms. In addition, we established the susceptibility of GluT, measured as [3H]-L-glutamate uptake in brain cortical prisms, to the inhibition of Na+/K(+)-ATPase by ouabain. Then, we tested the hypothesis that the Na+/K(+)-ATPase (measured as Rb+ uptake) can respond to changes in the activity of GluT produced by using GluT substrates as GluT-specific pharmacological tools. 3. The Na+/K(+)-ATPase inhibitor ouabain completely blocked Rb+ uptake (IC50 = 17 micromol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-L-glutamate uptake was eliminated; IC50 < 1 micromol/L). 4. None of the most commonly used GluT substrates and inhibitors, such as L-aspartate, D-aspartate, L-CCG III and LtPDC (all at 500 micromol/L), produced any significant changes in Rb+ uptake. 5. The N-methyl-D-aspartate (NMDA) receptor agonists (R,S)-(tetrazol-5-yl)-glycine and NMDA decreased Rb+ uptake in a manner compatible with their known neurotoxic actions. 6. None of the agonists or antagonists for any of the other major classes of glutamate receptors caused significant changes in Rb+ uptake. 7. We conclude that, even if a subpopulation of glutamate transporters in the rat cerebral cortex may be intimately linked to a fraction of Na+/K(+)-ATPase, it is not possible, under the present experimental conditions, to detect regulation of Na+/K(+)-ATPase by GluT.     

Ribavirin uptake by cultured human choriocarcinoma (BeWo) cells and Xenopus laevis oocytes expressing recombinant plasma membrane human nucleoside transporters

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.     

3-Methylcholanthrene increases phorbol 12-myristate 13-acetate-induced respiratory burst activity and intracellular calcium levels in common carp (Cyprinus carpio L) macrophages

Oral administration of triphenyltin chloride (TPT) (60 mg/kg body wt) inhibits insulin secretion by perturbing the cytoplasmic Ca(2+) concentration ([Ca(2+)]i) in pancreatic beta-cells of the hamster. To test the possibility that the abnormal levels of [Ca(2+)]i induced by TPT administration could be due to a defect in the cytoplasmic Na(+) concentration ([Na(+)]i) in the beta-cells, we investigated the effects of TPT administration on the changes of [Na(+)]i and [Ca(2+)]i induced by glucose or acetylcholine (ACh) and on the [Na(+)]i induced by ouabain, a potent inhibitor of Na(+),K(+)-ATPase. The changes of [Na(+)]i and [Ca(2+)]i were measured in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. TPT administration strongly reduced the rise in [Ca(2+)]i induced by 15 mM glucose with and without extracellular 135 mM Na(+). TPT administration also significantly reduced the rise of [Ca(2+)]i by 100 microM ACh in the presence of 5.5 or 15 mM glucose but not the amplitude of [Ca(2+)]i by 100 microM ACh in Na(+)-free medium. TPT administration attenuated the rise in [Na(+)]i induced by 100 microM ACh in the presence of either 3 or 5.5 mM glucose. However, TPT administration did not impair the [Na(+)]i in the presence of glucose (3, 5.5, and 15 mM) or of 100 microM ouabain with 3 mM glucose. TPT administration significantly suppressed the insulin secretion induced by 15 and 27.8 mM glucose or 100 microM ACh in the presence of 5.5 mM glucose. Our study suggests that triphenyltin has inhibitory effects on the cellular Ca(2+) response due to a reduction of [Ca(2+)]i after Na(+)-dependent and Na(+)-independent depolarization in islet cells of the hamster.     

Chlordecone impairs Na(+)-stimulated L-[3H]glutamate transport and mobility of 16-doxyl stearate in rat liver plasma membrane vesicles.

Chlordecone (CD) treatment of rat liver plasma membranes (LPM) provided in vitro evidence for mechanisms of in vivo liver dysfunction caused by CD. LPM preparations enriched 14- to 19-fold in the bile canalicular markers gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were isolated from male Sprague-Dawley rats. CD inhibited the bile canalicular-specific active transport of Na(+)-stimulated L-[3H]glutamate in LPM vesicles. CD (0.08 and 0.5 mumol/mg protein) reduced both the initial velocity and the maximum level of Na(+)-stimulated L-[3H]glutamate uptake without significantly reducing Na(+)-independent uptake. In vitro treatment of LPM with CD (0.2-1.0 mumols/mg protein) also reduced the mobility of a 16-doxyl stearate spin label probe in a concentration-dependent manner. No change in mobility was apparent at CD concentrations below 0.2 mumol/mg protein. These results demonstrated that CD impaired a bile canalicular-specific transport system and induced liver plasma membrane perturbation. Na(+)-stimulated L-[3H]glutamate uptake was more sensitive to CD than was detectable immobilization of the spin label probe.     

Tryptophan inhibits the [3H]glutamate uptake into Xenopus oocytes injected with rat brain mRNA.

We characterized the glutamate (Glu) uptake in Xenopus oocytes injected with rat brain mRNA. The Glu uptake into oocytes was higher in mRNA-injected oocytes than in vehicle-injected ones. Na+ omission or addition of tryptophan inhibited the uptake in mRNA-injected oocytes, although it did not affect that in vehicle-injected oocytes. These results suggest that Glu transporters with a tryptophan sensitivity different from that of Glu transporters in native oocytes are expressed after injection of rat brain mRNA.     

Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro

Scorpion neurotoxins targeting the Na(v) channel can be classified into two classes: alpha- and beta-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its alpha-neurotoxin TsTX-V on gamma-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect (3)H-Glu uptake. On the other hand, Ts venom (0.13 microg/mg) significantly inhibited both (3)H-GABA and (3)H-DA uptake ( approximately 50%). TsTX-V showed IC(50) values of 9.37 microM and 22.2 microM for the inhibition of (3)H-GABA and (3)H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 microM), indicating the involvement of voltage-gated Na(+) channels in this process. In the absence of Ca(2+), and at low Ts venom concentrations, the reduction of (3)H-GABA uptake was not as marked as in the presence of Ca(2+). TsTX-V did not reduce (3)H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced (3)H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of (3)H-GABA and (3)H-DA uptake in a Ca(2+)-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na(+) channels.     

Quantitative analysis of inward and outward transport rates in cells stably expressing the cloned human serotonin transporter: inconsistencies with the hypothesis of facilitated exchange diffusion

Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin (5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 microM). Apparent K(m) values for PCA induced transport were 564 microM and about 7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular Na(+) concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake increased approximately 5-fold and the V(max) value remained unchanged. The K(m) value for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%. When efflux was studied at saturating prelabeling conditions of both substrates, PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50% reduction in the V(max) value of transport into the cell, MPP(+) was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion.     

Uptake mechanism of pitavastatin, a new inhibitor of HMG-CoA reductase, in rat hepatocytes

To understand the mechanism underlying the highly liver-selective distribution of pitavastatin, uptake experiments were performed using rat hepatocytes.The uptake of pitavastatin into rat hepatocytes is carrier-mediated and involved nonspecific diffusion in the presence of Na(+). The michaelis constant (K(m)) was 26.0 micromol/L, maximal uptake velocity (V(max)) was 3124 pmol/min/mg protein, and non-specific uptake (P(dif)) was 1.16 microL/min/mg protein. There were no remarkable differences in these kinetic parameters between the presence and absence of Na(+).Experiments using metabolic inhibitors revealed that energy-dependent systems contribute to the uptake of pitavastatin in the liver. Some organic anions reduced the uptake into rat hepatocytes in a concentration-dependent manner. The observed rates of inhibition of pitavastatin uptake by BSP, TCA and pravastatin were compared with the predicted rates. The predicted values were calculated, assuming that BSP, TCA and pravastatin inhibit the uptake of pitavastatin in a competitive manner. The observed inhibition by BSP and TCA was similar to that predicted, but the observed inhibition by pravastatin was considerably less than that predicted.In conclusion, most of the pitavastatin taken up into the liver is transported by multiple carrier-mediated transporters such as Na(+)-independent multispecific anion transporters and energy-dependent transporters. In addition, these systems for pitavastatin may have features in common with the BSP and TCA transport system, and may partially involve the pravastatin transport system.     

Chronic haloperidol treatment impairs glutamate transport in the rat striatum.

High-affinity, Na(+)-dependent transport of glutamate into neurons and glial cells maintains the extracellular concentration of this neurotransmitter at a sub-toxic level. Chronic blockade of dopamine D(2) receptors with haloperidol elevates extracellular glutamate levels in the striatum. The present study examines the effect of long-term haloperidol treatment on glutamate transporter activity using an assay based on measuring the uptake of D-[3H]aspartate in striatal synaptosomes prepared from male Wistar rats. The maximal rate of glutamate transport in the striatum is reduced by 63% following 27 weeks of haloperidol treatment. This impairment of glutamate transport may be important in chronic neuroleptic drug action.     

Suppression of K(+)-induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium-derived hyperpolarizing factor

In intact mesenteric arteries, increasing [K(+)]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 microM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K(+)]o. In endothelium-denuded vessels, increasing [K(+)]o by 5 mM hyperpolarized the smooth muscle but K(+) had no effect after depolarization by 10 microM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K(+) was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K(+)-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K(+) via smooth muscle K(+) channels produces a local increase in [K(+)]o which impairs repolarization to added K(+). Thus, studies involving vessels precontracted with agonists which increase [K(+)]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K(+).     

The mode of inhibition of the Na+-K+ pump activity in mast cells by calcium.

1 The inhibition by calcium of the Na(+)-K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure populations of the cells by measuring the ouabain-sensitive uptake of the radioactive potassium analogue, 86rubidium (86Rb+). 2 Exposure of the cells to calcium induced a time- and concentration-dependent decrease in the ouabain-sensitive K+(86Rb+)-uptake of the cells without influencing the ouabain-resistant uptake. The development of the inhibition required the presence of potassium in the medium in the millimolar range (1.5-8.0 mM), and it did not occur at a concentration of potassium (0.24 mM) that is probably rate limiting for the pump activity. In the presence of 1 mM calcium full inhibition developed almost immediately and was not readily reversed. The inhibition was not significantly reduced by 15 min incubation with 1.2 mM EGTA. 3 The inhibitory action of calcium did not develop when the mast cells were incubated in a potassium-free medium, which is known to block Na(+)-K+ pump activity and allow accumulation of sodium inside the cells. Likewise, increasing the sodium permeability of the plasma membrane by monensin abolished the inhibition of the pump activity. In both cases, incubation of the cells with 4.7 mM potassium and tracer amounts of 86Rb+ resulted in a very large uptake of K+ (86Rb+) into the cells (up to 2 nmol per 10(6) cells min-1), indicating a high activity of the Na(+)-K+ pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotransmitter transporters: molecular function of important drug targets

The concentration of neurotransmitters in the extracellular space is tightly controlled by distinct classes of membrane transport proteins. This review focuses on the molecular function of two major classes of neurotransmitter transporter that are present in the cell membrane of neurons and/or glial cells: the solute carrier (SLC)1 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of glutamate, and the SLC6 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of dopamine, 5-HT, norepinephrine, glycine and GABA. Recent research has provided substantial insight into the structure and function of these transporters. In particular, the recent crystallizations of bacterial homologs are of the utmost importance, enabling the first reliable structural models of the mammalian neurotransmitter transporters to be generated. These models should be an important tool for developing specific drugs that, through selective interaction with transporters, could improve the treatment of serious neurological and psychiatric disorders.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

OATP1B1, OATP1B3, and mrp2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin II blocker.

Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (K(m)) of 29.3 +/- 9.9 microM. Both Na(+)-dependent and Na(+)-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na(+)-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with K(m) values of 42.6 +/- 28.6 and 71.8 +/- 21.6 microM, respectively. The Na(+)-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.     

Uptake kinetics and ion requirements for extraneuronal uptake of noradrenaline by arterial smooth muscle and collagen

1. The ionic requirements for noradrenaline uptake into vascular smooth muscle cells were studied by perfusing rabbit isolated ear arteries with noradrenaline (10(-3)M) either in Krebs solution or in Krebs solution modified by altering the concentration of one or more ion. Noradrenaline uptake was measured by quantitative microphotometry.2. Some uptake into smooth muscle continued in isotonic sucrose in the absence of all ions. Omission of Na(+) from the Krebs solution partially inhibited uptake as did high (100 mM) K(+). Omission of K(+), Ca(++) or Mg(++) had no effect on uptake. Lithium was able completely to substitute for Na(+).3. Alteration in ion concentration did not affect the binding of noradrenaline to collagen.4. The kinetics of uptake of noradrenaline into smooth muscle were analysed and found to be saturable with a Km of 4.9 x 10(-4)M.5. It is concluded that the ionic requirements of the transport mechanism for the uptake of noradrenaline by vascular smooth muscle show a relatively low specificity.