Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

Interaction of chlorpromazine with phospholipid membranes

The interaction of chlorpromazine (CPZ) with artificial membranes (egg-yolk phosphatidylcholine liposomes) has been studied. Measurements of the surface electric potential, which is modified in the presence of the ionized form of the drug, were obtained by electron paramagnetic resonance spectroscopy (EPR) using a positively charged amphiphilic spin-probe. This probe partitions between the aqueous and lipidic phases depending on the surface potential and on the structural state of the membrane. The surface potential was measured as a function of drug concentration in the range where the spectral line-shapes are not affected by the incorporation of the drug. From these experimental results and through an appropriate formalism we obtain information on the binding of the drug to the lipid bilayer and on the ionization of the drug in the lipidic phase. Correspondence to: C. Anteneodo     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio)

The environmental fate and ecotoxicological effect of pharmaceuticals are poorly understood, and standardized tests to detect and evaluate their potential effects in the environment are not available. We developed a zebrafish brain-specific microarray containing 682 neurologically relevant cDNA-fragments. To investigate the applicability of this microarray for studying neurotoxic modes-of-action and impact assessment of neuro-active pharmaceuticals in zebrafish, chlorpromazine was used as a model compound. After exposure to chlorpromazine (75 μg/L) for 2, 4, 14 and 28 days or control treatment RNA was extracted from brains of males and females. Fluorescently labeled cDNA was prepared and hybridized to the custom microarray. In total, 56 genes were differentially expressed in brains of male and/or female zebrafish, of which most genes were down-regulated. A clear difference in response to chlorpromazine exposure between males and females was observed with exposure time as well as in functional classes of affected genes. The presented study is one of the first reports on molecular effects of human neuro-active pharmaceuticals in aquatic non-target organisms. This new genomic tool successfully detected gene expression effects of exposure to chlorpromazine in the brain of zebrafish. Reported gene expression effects are found to be consistent with literature data for other laboratory animals.     

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31-0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 +/- 0.1 microM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate-glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nomega-nitro-L-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.     

Human cells and cell membrane molecular models are affected in vitro by chlorpromazine

This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 μM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 °C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer.     

Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers

Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK_a of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 °C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.     

The effect of chlorpromazine and tetracaine on the rapid axonal transport of neurosecretory material in the hypothalamo-neurohypophysial system of the rat

Summary The effects of chlorpromazine (cpz) and tetracaine (tc) on the rapid axonal transport of neurosecretory material (NSM) in the hypothalamo-neurohypophysial system was investigated. Following subarachnoidal injection of these drugs, the incorporation of (³⁵S) cysteine into proteins of the supraoptic nucleus was slightly depressed. The protein-bound radioactivity in the posterior pituitary was markedly lowered in experimental rats which indicates a partial blockage of the rapid axonal transport of NSM along the hypothalamoneurohypophysial tract. Both cpz and tc induced an increase in the number of mitochondria and profiles of granular endoplasmic reticulum. The axons in the infundibulum and neurohypophysis were enlarged by dammed organelles, indicating a blockage of axonal transport. There was an increased number of microvesicles, often arranged in a crystalloid pattern, in the terminals. The number and distribution of neurotubuli and neurofilaments were not changed. A possible stimulatory effect of cpz on the release of NSM from the neural lobe is assumed Possible mechanisms for the action of mitotic inhibitors, transquilizers and local anesthetics are discussed.The present work was supported by grants from Svenska Livforsäkringsbolags Fond, H. Hiertas stiftelse, Magnus Bergwalls stiftelse, The Swedish Medical Research Council No. B73-12X-2543-05B, Statens naturvetenskapliga forskningsråd No. 2535-8 and from the Medical Faculty, University of Göteborg. We are indebted to Mrs. Margareta Andersson, Mrs. Wally Holmberg, Mrs. Elisabeth Norström and Mrs. Ulla Svedin for excellent technical assistance, and to Miss Gull Grönstedt for careful secretarial work.     

The Neuroleptic Chlorpromazine Inhibits the Cationic and Stimulates the Anionic Phospholipid Precursor Synthesis in Human Lymphocytes

The widely used neuroleptic drug chlorpromazine (CPZ) influences membrane functions at the levels of ionic channels and receptors as shown. Here we show the effect of short term treatments by CPZ (30 μM), on the nucleotide-containing phospholipid precursors in human lymphocyte primary cultures. During 60 minutes incubation of the cells, the CDP-ethanolamine (CDP-EA) content was only slightly reduced (87 to 76 pmol/10⁶ cells), the amount of CDP-choline (CDP-Ch) was inhibited totally (from 25 to 0 pmol) upon the treatment with 30 μM CPZ under the same conditions. It has been shown earlier, that dCTP can be used as well as CTP for biosynthesis of phospholipids. Thus, the separation of the corresponding ribo- and deoxyribo-liponucleotides was developed. CPZ almost completely inhibited the synthesis of both dCDP-EA and dCDP-Ch under the same conditions The synthesis of the activated liponucleotide precursors, can be measured by incorporation of extracellular 14C-dCyt into both dCDP-EA and dCDP-Ch, as shown earlier. While the cationic deoxyribo-liponucleotide content (dCDP-Ch, dCDP-EA) was decreased, the labelling of the anionic phospholipid precursor dCDP-diacylglycerol (dCDP-DAG) was enhanced several times, it could be labelled only in the presence of CPZ from 14C-dCyd. Thus, a principal disturbance of the membrane phospholipid synthesis is presented (i.e., inhibition of the cationic and enhancement of the anionic dCDP-DAG synthesis). This profound influence on the membrane phospholipids by chlorpromazine, might be the primary effect that contributes to the wide spectrum of CPZ effects on neuronal cells.