Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.     

Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport

It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (K_p) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The K_p for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The K_p for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.     

A role for hepatic scavenger receptor class B, type I in decreasing high density lipoprotein levels in mice that lack phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt(+/+) and Pemt(-/-) mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt(-/-) mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt(+/+) mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt(-/-) compared with Pemt(+/+) livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [(3)H]cholesteryl ethers and [(3)H]PC by hepatocytes derived from Pemt(-/-) compared with Pemt(+/+) mice. Furthermore, we observed an increased association of [(3)H]cholesteryl ethers in livers of Pemt(-/-) compared with Pemt(+/+) mice after tail vein injection of [(3)H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI.     

Direct evidence of abca1-mediated efflux of cholesterol at the mouse blood�Cbrain barrier

We investigated the expression and function of Abca1 in wild-type C57BL/6, abca1(+/+), and abca1(-/-) mice brain capillaries forming the blood-brain barrier (BBB). We first demonstrated by quantitative RT-PCR and Western immunoblot that Abca1 was expressed and enriched in the wild-type mouse brain capillaries. In abca1(-/-) mice, we reported that the lack of Abca1 resulted in an 1.6-fold increase of the Abcg4 expression level compared to abca1(+/+) mice. Next, using the in situ brain perfusion technique, we showed that the [(3)H]cholesterol brain uptake clearance (Cl(up), μl/s/g brain), was significantly increased (107%) in abca1(-/-) mice compared to abca1(+/+) mice, meaning that the deficiency of Abca1 conducted to a significant decrease of the cholesterol efflux at the BBB level. In addition, the co-perfusion of probucol (Abca1 inhibitor) with [(3)H]cholesterol resulted in an increase of [(3)H]cholesterol Cl(up) (115%) in abca1(+/+) but not in abca1(-/-) mice, meaning that probucol inhibited selectively the efflux function of Abca1. In conclusion, our results demonstrated that Abca1 was expressed in the mouse brain capillaries and that Abca1 functions as an efflux transporter through the mouse BBB.     

Lipophilic cations: a group of model substrates for the multidrug-resistance transporter.

The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.     

Modulation of p-glycoprotein function by caveolin-1 phosphorylation

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.     

beta-Amyloid efflux mediated by p-glycoprotein

A large body of evidence suggests that an increase in the brain beta-amyloid (Abeta) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Abeta, however, less is known regarding its secretion from cells. We now report that p-glycoprotein (p-gp), an ATP-binding cassette (ABC) transporter, is an Abeta efflux pump. Pharmacological blockade of p-gp rapidly decrease extracellular levels of Abeta secretion. In vitro binding studies showed that addition of synthetic human Abeta1-40 and Abeta1-42 peptides to hamster mdr1-enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Abeta peptides across the plasma membranes of p-gp enriched vesicles, and showed that this phenomenon was both ATP- and p-gp-dependent. Taken together, our study suggests a novel mechanism of Abeta detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.     

Antinociceptive effects of morphine-6-glucuronide in homozygous MDR1a P-glycoprotein knockout and in wildtype mice in the hotplate test

Morphine-6-glucuronide (M6G), a major metabolite of morphine with agonist opioid-receptor activity, was reported to be a substrate of P-glycoprotein (P-gp). Inhibition of P-gp may thus result in higher brain uptake of M6G. The goal of this observer-blinded, placebo controlled study, was to compare the antinociceptive effects of M6G in homozygous P-gp knockout (mdr1a(-/-)) and wildtype (mdr1a(+/+)) mice. M6G was injected intraperitoneally as a single dose of 0, 0.5, 1, 2.5, 5, and 10 mg/kg. Eight P-gp knockout and eight wildtype mice were studied per dose. A hot plate test was performed before and 5, 15, 30, 60, 90, 120, and 150 min after M6G administration. Plasma-concentrations of M6G, morphine, and morphine-3-glucuronide (M3G) were measured after intraperitoneal injection of 5 mg/kg M6G in another 14 P-gp knockout and 14 wildtype mice. No difference neither in the dose response relationship, nor in the time course of response latency times were observed between P-gp knockout and wildtype mice. However, latency times increased with higher doses of M6G, with antinociception significantly different from placebo at a M6G dose of 5 and 10 mg/kg. P-gp knockout mice tended to have higher plasma concentrations than the wildtype. However, plasma concentrations widely overlapped between groups and therefore no statistical significant group difference could be detected. We conclude that despite reported doubling of M6G brain uptake, absence of mdr1a coded P-gp does not enhance antinociceptive effects of M6G in the hotplate test after acute single-dose administration in mdr1a(-/-) knockout mice.     

Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia

N-methyl- D -aspartate receptors (NMDARs) play a pivotal role in excitatory neurotransmission, synaptic plasticity and brain development. Clinical and experimental evidence suggests a dysregulation of NMDAR function and glutamatergic pathways in the pathophysiology of schizophrenia. We evaluated electrophysiological and behavioral properties of NMDAR deficiency utilizing mice that express only 5–10% of the normal level of NMDAR NR1 subunit. Auditory and visual event related potentials yielded significantly increased amplitudes for the P20 and N40 components in NMDAR deficient (NR1^neo−/−) mice suggesting decreased inhibitory tone. Compared to wild types, NR1^neo−/− mice spent less time in social interactions and showed reduced nest building. NR1^neo−/− mice displayed a preference for open arms of a zero maze and central zone of an open field, possibly reflecting decreased anxiety-related behavioral inhibition. However, locomotor activity did not differ between groups in either home cage environment or during behavioral testing. NR1^neo−/− mice displayed hyperactivity only when placed in a large unfamiliar environment, suggesting that neither increased anxiety nor non-specific motor activation accounts for differential behavioral patterns. Data suggest that NMDAR NR1 deficiency causes disinhibition in sensory processing as well as reduced behavioral inhibition and impaired social interactions. The behavioral signature in NR1^neo−/− mice supports the impact of impaired NMDAR function in a mouse model with possible relevance to negative symptoms in schizophrenia.     

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.     

Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m displayed statistically significant inhibition of food intake in an agonist-induced food intake model in SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (?/?) mice compared to mdr1a (+/+) mice after oral administration of 7m.     

An ALS mouse model with a permeable blood-brain barrier benefits from systemic cyclosporine A treatment

To test potentially beneficial drugs to amyotrophic lateral sclerosis (ALS), we created an ALS mouse model with a permeable blood-brain barrier, by crossing the G93A-SOD1 transgenic mouse with a multiple drug resistance type 1a/b (mdr1a/b) gene knockout mouse. To validate the model, we administered cyclosporine A intraperitoneally to the mice. Cyclosporine A accumulated in the brain and spinal cord of this mouse model, whereas it was unable to penetrate the CNS of mdr1a/b wild-type animals. Systemic administration of cyclosporine A extended the life of the double-mutant male mice by approximately 12%. Surprisingly, the effect was more robust in male mice and only marginal in female mice. These results demonstrate the usefulness of this combined mouse model for the testing of potentially therapeutic drugs and support the role of mitochondrial-mediated apoptosis in the pathway to motor neuron death in SOD1-associated ALS.     

Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice

P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [(3)H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (～3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (～2-fold, P < 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 × 10(9) colony-forming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2- to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

The alpha 3 subunit gene of the nicotinic acetylcholine receptor is a candidate gene for ethanol stimulation

Alcohol and nicotine are coabused, and preclinical and clinical data suggest that common genes may influence responses to both drugs. A gene in a region of mouse chromosome 9 that includes a cluster of three nicotinic acetylcholine receptor (nAChR) subunit genes influences the locomotor stimulant response to ethanol. The current studies first used congenic mice to confirm the influential gene on chromosome 9. Congenic F₂ mice were then used to more finely map the location. Gene expression of the three subunit genes was quantified in strains of mice that differ in response to ethanol. Finally, the locomotor response to ethanol was examined in mice heterozygous for a null mutation of the α3 nAChR subunit gene ( Chrna3 ). Congenic data indicate that a gene on chromosome 9, within a 46 cM region that contains the cluster of nAChR subunit genes, accounts for 41% of the genetic variation in the stimulant response to ethanol. Greater expression of Chrna3 was found in whole brain and dissected brain regions relevant to locomotor behavior in mice that were less sensitive to ethanol-induced stimulation compared to mice that were robustly stimulated; the other two nAChR subunit genes in the gene cluster (α5 and β4) were not differentially expressed. Locomotor stimulation was not expressed on the genetic background of Chrna3 heterozygous (+/−) and wild-type (+/+) mice; +/− mice were more sensitive than +/+ mice to the locomotor depressant effects of ethanol. Chrna3 is a candidate gene for the acute locomotor stimulant response to ethanol that deserves further examination.     

Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice.

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (-/-) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [(13)C]-linoleic acid absorption was determined by measuring [(13)C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [(13)C]-linoleic acid at 2 h after enteral administration was significantly lower in (-/-) compared to (+/+) mice (P相似文献   

Co-amplification and over-expression of two mdr genes in a multidrug-resistant human colon carcinoma cell line.

The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.