Showing papers in "Molecular Pharmacology in 1997"

Neuroprotection against Oxidative Stress by Estrogens: Structure-Activity Relationship

Abstract: Oxidative stress-induced neuronal cell death has been implicated in different neurological disorders and neurodegenerative diseases; one such ailment is Alzheimer’s disease. Using the Alzheimer’s disease-associated amyloid β protein, glutamate, hydrogen peroxide, and buthionine sulfoximine, we investigated the neuroprotective potential of estrogen against oxidative stress-induced cell death. We show that 17-β-estradiol, its nonestrogenic stereoisomer, 17-α-estradiol, and some estradiol derivatives can prevent intracellular peroxide accumulation and, ultimately, the degeneration of primary neurons, clonal hippocampal cells, and cells in organotypic hippocampal slices. The neuroprotective antioxidant activity of estrogens is dependent on the presence of the hydroxyl group in the C3 position on the A ring of the steroid molecule but is independent of an activation of estrogen receptors.

(-)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB.

Abstract: Nitric oxide (NO) plays an important role in inflammation and multiple stages of carcinogenesis. We investigated the effect of various tea polyphenols and caffeine on the induction of NO synthase (NOS) in thioglycollate-elicited and lipopolysaccharide (LPS)-activated peritoneal macrophages. Gallic acid (GA), (-)-epigallocatechin (EGC), and (-)-epigallocatechin-3-gallate (EGCG), the major tea catechin, were found to inhibit inducible NOS (iNOS) protein in activated macrophages. EGCG, a potent antitumor agent with anti-inflammatory and antioxidant properties, inhibited NO generation, as measured by the amount of nitrite released into the culture medium. Inhibition of NO production was observed when cells were cotreated with EGCG and LPS. iNOS activity in soluble extracts of lipopolysaccharide-activated macrophages treated with EGCG (5 and 10 microM) for 6-24 hr was significantly lower than that in macrophages without EGCG treatment. Western blot, reverse transcription-polymerase chain reaction, and Northern blot analyses demonstrated that significantly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS were expressed in lipopolysaccharide-activated macrophages with EGCG compared with those without EGCG. Electrophoretic mobility shift assay indicated that EGCG blocked the activation of nuclear factor-kappaB, a transcription factor necessary for iNOS induction. EGCG also blocked disappearance of inhibitor kappaB from cytosolic fraction. These results suggest that EGCG decreases the activity and protein levels of iNOS by reducing the expression of iNOS mRNA and the reduction could occur through prevention of the binding of nuclear factor-kappaB to the iNOS promoter, thereby inhibiting the induction of iNOS transcription.

High affinity glutamate transporters: regulation of expression and activity.

Abstract: L-Glutamic acid is a major excitatory neurotransmitter in the mammalian central nervous system. The termination of the glutamatergic transmission and the clearance of the excessive, neurotoxic concentrations of glutamate is ensured by a high affinity glutamate uptake system. Four homologous types of Na/K-dependent high affinity glutamate transporters, glutamate/aspartate transporter, glutamate transporter 1, excitatory amino acid carrier 1, and excitatory amino acid transporter 4, have recently been cloned and were assigned to a separate gene family, together with two neutral amino acid carriers, alanine/serine/cysteine transporter 1/serine/alanine/threonine transporter and adipocyte amino acid transporter. The genomic organization of these transporters is still under investigation. Very little is known about the nature of the factors and molecular mechanisms that regulate developmental, regional, and cell type-specific expression of the glutamate transporters and their aberrant functioning in neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and Alzheimer's disease). Some experimental conditions (e.g., ischemia, corticostriatal lesions, hyperosmolarity, culturing conditions) and several naturally occurring and synthetic compounds (e.g., glutamate receptor agonists, dopamine, alpha1- and beta-adrenergic agonists, cAMP, phorbol esters, arachidonic acid, nitric oxide, oxygen free radicals, amyloid beta-peptide, tumor necrosis factor-alpha, glucocorticosteroids, unidentified neuronal factors) affect the molecular expression and activity of glutamate transporters. Further elucidation of the molecular events that link epigenetic signals with transcriptional and post-transcriptional mechanisms (e.g., alternative splicing, translation and post-translational modifications) is crucial for the development of selective pharmacological tools and strategies interfering with the expression of the individual glutamate transporters.

Cloning and Functional Expression of a Human Liver Organic Cation Transporter

Abstract: Polyspecific organic cation transporters in the liver mediate the elimination of a wide array of endogenous amines and xenobiotics. In contrast to our understanding of the mechanisms of organic cation transport in rat liver, little is known about the mechanisms of organic cation transport in the human liver. We report the cloning, sequencing, and functional characterization of the first human polyspecific organic cation transporter from liver (hOCT1). hOCT1 (554 amino acids) is 78% identical to the previously cloned organic cation transporter from rat, rOCT1 [Nature (Lond.) 372:549–552 (1994)]. InXenopus laevis oocytes injected with the cRNA of hOCT1, the specific uptake of the organic cation3H-1-methyl-4-phenylpyridinium (3H-MPP+) was significantly enhanced (8-fold) over that in water-injected oocytes. Uptake of3H-MPP+ was saturable (Km = 14.6 ± 4.39 μm) and sensitive to membrane potential. Both small monovalent organic cations such as tetraethylammonium andN1-methylnicotinamide and bulkier organic cations (e.g., vecuronium and decynium-22) inhibited the uptake of3H-MPP+. In addition, the bile acid taurocholate inhibited the uptake of 3H-MPP+ in oocytes expressing hOCT1. Northern analysis demonstrated that the mRNA transcript of hOCT1 is expressed primarily in the human liver, whereas the mRNA transcript of rOCT1 is found in rat kidney, liver, intestine, and colon [Nature (Lond.) 372:549–552 (1994)]. In comparison to rOCT1, hOCT1 exhibits notable differences in its kinetic characteristics and tissue distribution. The functional expression of hOCT1 will provide a powerful tool for elucidation of the mechanisms of organic cation transport in the human liver and understanding of the mechanisms involved in the disposition and hepatotoxicity of drugs.

The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B).

Abstract: A monoclonal antibody (R1JHL) against the NR1 subunit of theN-methyl-d-aspartate (NMDA) receptor has been developed that recognizes an epitope in the region of the amino-terminal amino acids 341–561 (a region common to all splice variants of NR1). This monoclonal antibody identifies a broad band at 115 kDa in immunoblots using membranes from NR1-transfected cells and from rat brain tissue. No cross-reactivity with any NR2 subunit is seen. With the goal to determine quantitatively the subunit composition of cortical NMDA receptors, we used the monoclonal antibody to NR1 and polyclonal antibodies against the NR2A and NR2B subunits to perform immunoprecipitations of receptor subunits from solubilized adult rat cortical membranes. Solubilization of the receptor subunits was accomplished under both nondenaturing (native) conditions, under which the subunits seem to remain associated with one another, and denaturing conditions, under which the subunits are dissociated from each other. Although each of these antibodies selectively immunoprecipitates only its corresponding (cognate) subunit when the subunits have been solubilized under denaturing conditions, each of the antibodies immunoprecipitates a sizable fraction of the other two NMDA receptor subunits when membranes are solubilized under nondenaturing conditions, indicating an interaction in situ. Using quantitative immunoblot analysis of the three subunits in both the pellets and supernatants from the immunoprecipitations, we found 1) the dominant NMDA receptor complex in adult rat cortex contains at least three subunits, NR1/NR2A/NR2B; 2) a smaller fraction of NMDA receptors are composed of only two subunits, NR1/NR2B or NR1/NR2A; 3) there are no complexes that contain NR2A/NR2B that do not contain NR1; 4) only a small fraction of each subunit is not associated with any other NMDA receptor subunit; 5) no coimmunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical interaction between these NMDA receptor subunits and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subunits. These results suggest that functional studies with recombinant receptors composed of at least three subunits may be the most physiologically meaningful.

Expression of Enzymatically Active CYP3A4 by Caco-2 Cells Grown on Extracellular Matrix-Coated Permeable Supports in the Presence of 1α,25-Dihydroxyvitamin D3

Abstract: The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b5, liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (approximately 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.

Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue : identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue

Abstract: Family 1 UDP-glucuronosyltransferases (UGTs) (UGT1A) are encoded by a locus that predicts the existence of at least nine individual proteins. The different proteins are generated by exon-sharing, which results in the production of a family of proteins that contain identical, 245-amino acid, carboxyl-terminal domains and an amino-terminal region of approximately 280 amino acids. The diversity of the UGT1A locus suggests the existence of complex regulation, most likely designed to account for the variable and specific glucuronidation requirements. However, the tissue-specific and extrahepatic regulation of the complete UGT1A locus has not been defined to date. In this study, quantitative duplex reverse transcription-polymerase chain reaction was used to analyze UGT1A RNA expression in 16 hepatic, four biliary, and two gastric human tissue specimens. UGT1A3 and UGT1A6 were found to be expressed in the three tissues, whereas UGT1A5 and UGT1A8 were not expressed. Hepatocellular and biliary tissue expressed UGT1A1 and UGT1A4 but hepatocellular tissue uniquely expressed UGT1A9, whereas biliary tissue expressed UGT1A10. In contrast to hepatocellular tissue, gastric tissue expressed UGT1A7 in addition to UGT1A10. The expression of UGT1A9 in hepatic tissue, UGT1A7 in gastric tissue, and UGT1A10 in biliary and gastric tissue provides evidence for the selective regulation of the UGT1A locus in hepatic and extrahepatic tissues. The newly identified UGT1A7 and UGT1A10 transcripts were cloned and found to be 95.86% identical. Sequence analysis confirmed two proteins with divergent amino termini of 285 residues and identical carboxyl termini of 245 residues. This study provides evidence for hepatic and extrahepatic regulation of the human UGT1A locus and identifies two novel extrahepatic transcripts of the UGT1A family.

Aryl hydrocarbon receptor-dependent induction of cyp1a1 by bilirubin in mouse hepatoma hepa 1c1c7 cells.

Abstract: Heme metabolism normally involves enzymatic conversion to biliverdin and subsequently to bilirubin, catalyzed by heme oxygenase and biliverdin reductase, respectively. We examined the ability of exogenously added hemin, biliverdin, or bilirubin to regulate Cyp1a1, an enzyme that may be active in bilirubin elimination. A substantial dose-dependent increase in Cyp1a1 mRNA occurred after treatment of Hepa 1c1c7 cells with either of the three compounds. This increase was readily apparent 1 hr after treatment with biliverdin or bilirubin but required >/=2 hr with hemin. Treatment of Hepa 1c1c7 cells with these compounds also caused a dose-dependent increase in Cyp1a1-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity. Of the three compounds, bilirubin produced the greatest maximal increase in Cyp1a1 mRNA and EROD (5.5-, 10.5-, and 15-fold for 100 microM hemin, biliverdin, and bilirubin, respectively) activity. The RNA polymerase inhibitor actinomycin D completely blocked Cyp1a1 induction by these compounds, indicating a requirement for de novo RNA synthesis via transcriptional activation. The protein synthesis inhibitor cycloheximide did not affect Cyp1a1 mRNA induction, indicating a lack of requirement for labile protein factors. In contrast, EROD induction by hemin, biliverdin, or bilirubin was completely blocked by cycloheximide treatment, indicating that the increase in enzyme activity is dependent on increased Cyp1a1 apoprotein synthesis. Aryl hydrocarbon receptor (AHR)- and AHR nuclear translocator-deficient mutant Hepa 1c1c7 cells did not exhibit increased Cyp1a1 mRNA or EROD activity after treatment with these compounds, indicating the requirement for a functional AHR for this response. Consistent with this, hemin, biliverdin, and bilirubin were able to induce expression of the dioxin-response element/luciferase reporter plasmid pGudLuc1.1 after transient transfection into wild-type Hepa 1c1c7 cells. Gel retardation assays demonstrated that bilirubin, but not hemin or biliverdin, was able to transform the AHR to a form capable of specifically binding to a 32P-labeled oligonucleotide containing a dioxin-response element sequence. These data indicate that bilirubin induces Cyp1a1 gene transcription through direct interaction with the AHR. In contrast, hemin and biliverdin seem to induce Cyp1a1 indirectly by serving as precursors to the endogenous formation of bilirubin via normal heme metabolism pathways. This is the first direct demonstration that the endogenous heme metabolite bilirubin can directly regulate Cyp1a1 gene expression and enzymatic activity in an AHR-dependent manner.

The Reduction of α-Tocopherolquinone by Human NAD(P)H:Quinone Oxidoreductase: The Role of α-Tocopherolhydroquinone as a Cellular Antioxidant

Abstract: α-Tocopherolquinone (TQ), a product of α-tocopherol oxidation, can function as an antioxidant after reduction to α-tocopherolhydroquinone (TQH2). We examined the ability of human NAD(P)H:quinone oxidoreductase (NQO1) to catalyze the reduction of TQ to TQH2 in cell-free and cellular systems. In reactions with purified human NQO1, TQ was reduced to TQH2. Kinetic parameters for the reduction of TQ by NQO1 (Km = 370 μm;kcat = 5.6 × 103 min−1;kcat/Km = 15 min−1 · μm−1) indicate that NQO1 can efficiently reduce TQ to TQH2. A comparison of the rate of reduction of TQ and coenzyme Q10 by NQO1 showed that TQ is reduced more efficiently than coenzyme Q10. Experiments with either Chinese hamster ovary (CHO) cells stably transfected with human NQO1 or CHO cell sonicates demonstrated a correlation between NQO1 activity and TQ reduction to TQH2. CHO cells with elevated NQO1 generated and maintained higher levels of TQH2 after treatment with TQ relative to NQO1-deficient CHO cells. TQH2generated from NQO1-mediated reduction of TQ prevented cumene hydroperoxide-induced lipid peroxidation in rat liver microsomes. In addition, cumene hydroperoxide-induced lipid peroxidation was inhibited more efficiently by TQ in CHO cell lines with elevated NQO1 activity. These data demonstrate that NQO1 can reduce TQ to TQH2 and that TQH2 can function as an efficient antioxidant. This work suggests that one of the physiological functions of NQO1 may be to regenerate antioxidant forms of α-tocopherol.

The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Polymers, Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel, and Inhibits the Growth of Paclitaxel-Resistant Cells

Abstract: The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.

Internal Trafficking and Surface Mobility of a Functionally Intact β2-Adrenergic Receptor-Green Fluorescent Protein Conjugate

Abstract: The β2-adrenergic receptor (β2AR) is prototypic of the large family of G protein-coupled receptors (GPCRs) whose desensitization and resensitization are regulated by intracellular kinases, arrestin proteins, phosphatases, and ill-defined components of the cellular endocytic machinery. The study of β2AR signal transduction and behavior in living cells is technically difficult because of the relatively low cellular expression of the receptor and a lack of useful biological reagents. Availability of a functional β2AR tagged with the highly sensitive Green Fluorescent Protein (GFP) could allow measurements of the various properties of the β2AR. We demonstrate that a fully functional β2AR/GFP can be engineered. In mammalian cells, β2AR/S65T/GFP demonstrates strong, diffuse plasma membrane fluorescence when observed with 480 nm excitation. The fluorescent receptor binds agonist and antagonist, stimulates adenylyl cyclase, undergoes phosphorylation, and is internalized in a manner indistinguishable from wild-type receptor. We then show that its internal trafficking and surface mobility can be determined by measuring only the endogenous fluorescence of the conjugate. β2AR/S65T/GFP was found to be localized on endosomal membranes in living cells within minutes of agonist treatment, and within 15 min it is observed in more complicated structures formed from fusion of multiple endosomes. Finally, its free diffusion (diffusion coefficient, 4.0–12 × 10−9 cm2/sec) was assessed on living cells using photobleaching recovery measurements. This approach and the fidelity of the biochemical properties of the β2AR/S65T/GFP demonstrate that real-time optical measurements of β2AR (as well as other GPCR) interactions and dynamics on living cells are feasible.

Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization.

Abstract: Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the beta-adrenergic receptor kinase (betaARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the beta2-adrenergic receptor (beta2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that betaARK and arrestin proteins (beta-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the beta2AR to be phosphorylated by betaARK or to interact normally with beta-arrestin substantially reduces agonist-mediated sequestration. To evaluate betaARK and beta-arrestin regulation of beta2AR sequestration, we examined the relationship between betaARK and/or beta-arrestin expression and beta2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous beta-arrestin expression and beta2AR sequestration, whereas HEK 293 had the highest. Overexpression of beta-arrestin, but not betaARK, in COS cells increased the extent of wild-type beta2AR sequestration to levels observed in HEK 293 cells. However, a betaARK phosphorylation-impaired beta2AR mutant (Y326A) required the simultaneous overexpression of both betaARK and beta-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of betaARK and beta-arrestin expression. Moreover, an agonist-mediated translocation of wild-type beta2AR and endogenous beta-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular betaARK and arrestin proteins synergistically regulate beta2AR sequestration but also that beta-arrestins directly regulate beta2AR trafficking as well as desensitization.

Characterization of recombinant human p2x4 receptor reveals pharmacological differences to the rat homologue

Abstract: We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X 4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X 4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X 4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X 4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embryonic kidney 293 cells transfected with human P2X 4 cDNA allowed us to determine the fraction of the current carried by Ca 2+ ; this was ∼8%, demonstrating a high Ca 2+ permeability. Low extracellular Zn 2+ concentrations (5–10 μm) increase the apparent gating efficiency of human P2X 4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (>100 μm) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X 4 receptor displays a very similar agonist potency profile to that of rat P2X 4 (ATP ≫ 2-methylthio-ATP ≥ CTP > α,β-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid.

P-glycoprotein substrates and antagonists cluster into two distinct groups.

Abstract: To gather further insight into the interaction between P-glycoprotein (Pgp) and its substrates, 167 compounds were analyzed in multidrug resistant human colon carcinoma cells. These compounds were selected from the National Cancer Institute Drug Screen repository using computer-generated correlations with known Pgp substrates and antagonists. The compounds were prospectively defined as Pgp substrates if cytotoxicity was increased > or =4-fold by the addition of cyclosporin A (CsA) and as Pgp antagonists if inhibition of efflux increased rhodamine accumulation by 4-fold. Among the 84 agents that met either criterion, 35 met only the criterion for substrates, 42 met only the criterion for antagonists, and only seven met both criteria. Thus, compounds interacting with Pgp form two distinct groups: one comprising cytotoxic compounds that are transported and have poor or no antagonistic activity and a second comprising compounds with antagonistic activity and no evidence of significant transport. Vinblastine accumulation and kinetic studies performed on a subset of 18 compounds similarly differentiated substrates and antagonists, but inhibition of 3H-azidopine labeling and induction of ATPase activity did not. These data support an emerging concept of Pgp in which multiple regions instead of specific sites are involved in drug transport.

Programmed Cell Death in Neurons: Focus on the Pathway of Nerve Growth Factor Deprivation-Induced Death of Sympathetic Neurons

Abstract: Extensive programmed cell death (PCD) occurs in the developing nervous system. Neuronal death occurs, at least in part, because neurons are produced in excess during development and compete with each other for the limited amounts of the survival-promoting trophic factors secreted by target tissues. Neuronal death is apoptotic and utilizes components that are conserved in other PCD pathways. In this review, we discuss the mechanism of trophic factor-dependent neuronal cell death by focusing on the pathway of nerve growth factor (NGF) deprivation-induced sympathetic neuronal death. We describe the biochemical and genetic events that occur in NGF-deprived sympathetic neurons undergoing PCD. Participation of the Bcl-2 family of proteins and the interleukin-1beta-converting enzyme family of proteases (caspases) in this and other models of neuronal death is also examined. The order and importance of these components during NGF deprivation-induced sympathetic neuronal death are discussed.

Distinct Sites for Inverse Modulation of N-Methyl-D-Aspartate Receptors by Sulfated Steroids

Abstract: Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.

Differential Sensitivity of RecombinantN-Methyl-d-Aspartate Receptor Subtypes to Zinc Inhibition

Abstract: Zinc has been shown to be present in synaptic vesicles of a subset of glutamatergic boutons and is believed to be coreleased with glutamate at these synapses. A variety of studies have suggested that zinc might play a role in modulation of excitatory transmission, as well as excitotoxicity, by inhibitingN-methyl-d-aspartate (NMDA)-type glutamate receptors. To further investigate the modulatory effects of zinc on NMDA receptors of different subunit compositions, we coexpressed the recombinant subunit NR1 with NR2A and/or NR2B in HEK 293 cells. In whole-cell patch-clamp recordings from these transfected cells, zinc inhibited peak glutamate-evoked current responses in a noncompetitive manner, but there were significant differences between the receptor subtypes in sensitivity to zinc inhibition. For NR1/NR2A, ∼40% of the peak current was inhibited by zinc in a voltage-independent manner with an IC50 value of 5.0 ± 1.6 nm and at a VH value of −60 mV; the remainder was blocked at a second, voltage-dependent site with an IC50 value of 79 ± 18 μm. In contrast, NR1/NR2B currents showed nearly complete inhibition at a voltage-independent site with an IC50 value of 9.5 ± 3.3 μm. Cells cotransfected with NR1, NR2A, and NR2B showed zinc sensitivity intermediate between that characteristic of NR1/NR2A and that of NR1/NR2B. Furthermore, zinc accelerated the macroscopic desensitization of both NR1/NR2A and NR1/NR2B in a dose-dependent manner, apparently independently of glycine-sensitive desensitization and Ca2+-dependent inactivation; maximal effects were to decrease desensitization time constants for NR1/NR2A by ∼75% and for NR1/NR2B by ∼90%. Differential modulation of NR1/NR2A and NR1/NR2B currents by zinc may play a role in regulating NMDA receptor-induced synaptic plasticity and neurotoxicity.

Cloning and Expression of Rat Metabotropic Glutamate Receptor 8 Reveals a Distinct Pharmacological Profile

Abstract: The metabotropic glutamate receptor (mGluR) cDNAs were originally cloned from rat, except for the mouse cDNA clone encoding mGluR8. Mouse mGluR8 couples weakly to the inhibition of adenylate cyclase, thus hindering the characterization of its pharmacological properties. We isolated a rat mGluR8 cDNA that encodes a protein of 908 amino acids.In situ hybridization revealed prominent mGluR8 mRNA expression in olfactory bulb, pontine gray, lateral reticular nucleus of the thalamus, and piriform cortex. Less abundant expression was detected in cerebral cortex, hippocampus, cerebellum, and mammillary body. Glutamate evoked pertussis toxin-sensitive potassium currents inXenopus laevis oocytes coexpressing mGluR8 and G protein-coupled inwardly rectifying potassium channels. mGluR8 was also activated by the group III-specific agonistl-2-amino-4-phosphonobutyric acid; (2(S),1′(S),2′(S)]-2-(carboxycyclopropyl)glycine, which has been frequently used as a selective group II agonist; and the nonselective agonist (1(S),3(R)]-1-aminocyclopentane-1,3-dicarboxylic acid but not by the group I-specific agonist 3,5-dihydroxyphenylglycine or the group II-specific agonist [2(S),1′(R),2′(R),3′(R)]-2-(2,3-dicarboxycyclopropyl)glycine. The agonist profile in order of potency was [2(S),1′(S),2′(S)]-2-(carboxycyclopropyl)glycine ≈ l-2-amino-4-phosphonobutyric acid > glutamate ≫ [1(S),3(R)]-1-aminocyclopentane-1,3-dicarboxylic acid, with EC50 values of 0.63, 0.67, 2.5, and 47 μm, respectively. Both the group I/II-specific antagonist (R,S)-α-methyl-4-carboxyphenylglycine and the group III-specific antagonist α-methyl-amino-phosphonobutyrate inhibited mGluR8. The pharmacological profile of mGluR8 is distinct among mGluRs but closely matches that of presynaptic inhibition in some central nervous system pathways. Thus, cellular responses mediated by both group II and III agonists may in some cases reflect activation of mGluR8 rather than multiple mGluR subtypes.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression

Abstract: 2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

μ-Opioid Receptor-Stimulated Guanosine-5′-O-(γ-thio)-triphosphate Binding in Rat Thalamus and Cultured Cell Lines: Signal Transduction Mechanisms Underlying Agonist Efficacy

Abstract: G protein activation by different μ-selective opioid agonists was examined in rat thalamus, SK-N-SH cells, and μ-opioid receptor-transfected mMOR-CHO cells using agonist-stimulated guanosine-5′-O-(γ-thio)-triphosphate ([35S]GTPγS) binding to membranes in the presence of excess GDP. [d-Ala2,N-MePhe4,Gly5-ol]Enkephalin (DAMGO) was the most efficacious agonist in rat thalamus and SK-N-SH cells, followed by (in rank order) fentanyl = morphine ≫ buprenorphine. In mMOR-CHO cells expressing a high density of μ receptors, no differences were observed among DAMGO, morphine or fentanyl, but these agonists were more efficacious than buprenorphine, which was more efficacious than levallorphan. In all three systems, efficacy differences were magnified by increasing GDP concentrations, indicating that the activity state of G proteins can affect agonist efficacy. Scatchard analysis of net agonist-stimulated [35S]GTPγS binding revealed two major components responsible for agonist efficacy differences. First, differences in theKD values of agonist-stimulated [35S]GTPγS binding between high efficacy agonists (DAMGO, fentanyl, and morphine) and classic partial agonists (buprenorphine and levallorphan) were observed in all three systems. Second, differences in the Bmax value of agonist-stimulated [35S]GTPγS binding were observed between DAMGO and morphine or fentanyl in rat thalamus and SK-N-SH cells and between the high efficacy agonists and buprenorphine or levallorphan in all three systems. These results suggest that μ-opioid agonist efficacy is determined by the magnitude of the receptor-mediated affinity shift in the binding of GTP (or [35S]GTPγS) versus GDP to the G protein and by the number of G proteins activated per occupied receptor.

Positive Cooperativity of Acetylcholine and Other Agonists with Allosteric Ligands on Muscarinic Acetylcholine Receptors

Abstract: It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M2 and brucine and carbachol on the M1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.

Canine Mast Cell Adenosine Receptors: Cloning and Expression of the A3 Receptor and Evidence that Degranulation Is Mediated by the A2B Receptor

Abstract: We cloned and characterized the canine A3 adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A3AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor.125I-N6-Aminobenzyladenosine binds to two affinity states of canine A3AR withKD values of 0.7 ± 0.1 and 16 ± 0.8 nm, reflecting G protein-coupled and -uncoupled receptors, respectively. Xanthine antagonists bind with similar affinities to human, canine, and rabbit receptors but with 80–400-fold lower affinities to rat A3AR. Although canine BR mastocytoma cells contain A1AR, A2BAR, and A3AR, degranulation seems to be mediated primarily by A2BARs stimulated by the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA) but not by the A3-selective agonistN6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (Ki = 7 μm), an antiasthmatic xanthine with low affinity (Ki > 100 μm) for A1AR, A2AAR, and A3AR. NECA increases canine mastocytoma cell cAMP, Ca2+, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7–15 μm enprofylline. The results suggest that (i) the cloned canine A3AR is structurally and pharmacologically more similar to human than to rat A3AR; (ii) the A2BAR, and not the A1AR or A3AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A2BAR couples to both Ca2+ mobilization and cAMP accumulation. Although A2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.

Modulation of G Protein-Coupled Receptors by an Estrogen Receptor that Activates Protein Kinase A

Abstract: 17β-Estradiol (E2) rapidly (<20 min) attenuates the ability of μ-opioids to hyperpolarize guinea pig hypothalamic (β-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E2. E2 acted stereospecifically with physiologically relevant concentration dependence (EC50 = 8 nm) to cause a 4-fold reduction in the potency of a μ-opioid agonist to activate an inwardly rectifying K+conductance. Using Schild analysis to estimate the affinity of the μ-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the μ-opioid receptor or alter the affinity of the μ receptor. Both the nonsteroidal estrogen diethylstilbestrol and the “pure” antiestrogen ICI 164,384 blocked the actions of E2, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the μ-opioid receptor from its K+ channel, implying a nongenomic mechanism of action by E2. The actions of E2 were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the μ-opioid receptor from its K+ channel. Because we have previously shown that γ-aminobutyric acidB receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.

Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels.

Abstract: Toxins isolated from scorpion, snake, and spider venoms are valuable tools to probe the physiologic function and structure of ion channels. In this study, we have isolated three new toxins (heteropodatoxins) from the venom of a spider, Heteropoda venatoria. These toxins are structurally similar peptides of 29 to 32 amino acids and share sequence homology with hanatoxins isolated from the venom of a Chilean tarantula. The heteropodatoxins prolonged the action-potential duration of isolated rat ventricular myocytes, suggesting that the peptides block K+ currents. The effect of toxins on cardiac K+ currents were studied using voltage clamp techniques. The toxins blocked the transient outward K+ current but not other K+ currents in isolated rat cardiac myocytes. The mechanism of block was studied further using Kv4.2, a cloned channel believed to underlie transient outward K+ current in rat myocytes. The toxins blocked Kv4.2 current expressed in Xenopus laevis oocytes in a voltage-dependent manner, with less block at more positive potentials. In addition, the toxins slowed the time course of current activation and inactivation and shifted the voltage dependence of current inactivation to more positive potentials. The heteropodatoxins represent new pharmacologic probes to study the role of Kv4.2 channels in cardiac and neural tissue.

ATP-dependent transport of aflatoxin B1 and its glutathione conjugates by the product of the multidrug resistance protein (MRP) gene.

Abstract: Glutathione-S-transferase-catalyzed conjugation of glutathione (GSH) to aflatoxin B1-8,9-epoxide plays an important role in preventing binding of this ultimate carcinogen to target macromolecules. Once formed, the aflatoxin B1-epoxide-GSH conjugates are actively extruded from the cell by an unidentified ATP-dependent export pump or pumps. Two possible candidates for this GSH conjugate pump are the 190-kDa multidrug resistance protein (MRP) and the 170-kDa P-glycoprotein. Both proteins belong to the ATP-binding cassette superfamily of transmembrane transport proteins and confer resistance to a similar spectrum of natural-product drugs. Using membrane vesicles from MRP-transfected cells, we found that MRP transports GSH conjugates of both the endo-isomers and exo-isomers of aflatoxin B1-8,9-epoxide in an ATP-dependent, osmotically sensitive manner (V(max) = 180 pmol/mg/min, K(m) = 189 nM). Membrane vesicles from P-glycoprotein-overexpressing cells showed very low levels of transport. MRP-mediated transport was inhibited by an MRP-specific monoclonal antibody and by a variety of GSH derivatives and cholestatic steroid glucuronides. ATP-dependent transport of unmodified aflatoxin B1 by MRP-enriched membrane vesicles was low but markedly enhanced in the presence of 5 mM GSH, even though GSH conjugates of aflatoxin B1 were not formed by the vesicles. These data demonstrate that MRP is capable of energy-dependent transport of aflatoxin B1 and its GSH conjugates and suggest a potential protective role for MRP in mammalian chemical carcinogenesis.

Antagonist-stimulated internalization of the G protein-coupled cholecystokinin receptor.

Abstract: Receptor-mediated endocytosis has been observed after agonist occupation of several G protein-coupled receptors, which contributes to the desensitization response to agonist stimulation; however, the cellular signals required to initiate this process are unclear. In this study, we developed and characterized a new antagonist analogue of cholecystokinin (d-Tyr-Gly-[(Nle28,31,d-Trp30)cholecystokinin-26–32]-phenethyl ester) that can be tagged with a fluorescent rhodamine and radioiodinated. This has permitted us to demonstrate that antagonist occupation of the cholecystokinin receptor also results in receptor internalization, which dissociates this response from second messenger signaling activities and receptor phosphorylation. Immunolocalization of this receptor after occupation with an established nonpeptidyl antagonist confirmed this phenomenon. Antagonist-induced receptor internalization probably results from stabilization of the receptor in a conformation that exposes a domain critical to directing it into the clathrin-dependent endocytic pathway. This work provides evidence for a new and independent mechanism for receptor internalization, provides a mechanism for the rarely observed phenomenon of antagonist-induced desensitization, and raises important issues regarding the approach to establish optimal treatment regimens for antagonist drugs.

Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity.

Abstract: Genistein (5,7,4′-trihydroxyisoflavone), an isoflavinoid found in soy beans, has been identified as potentially causal for the low incidence of metastatic prostate cancer (PCa) in certain countries. Although genistein-induced PCa cell adhesion has been identified as a possible causative mechanism, direct growth inhibition by genistein has been reported and also could be causal. If in vivo growth inhibition was significant, then growth inhibition should occur at concentrations attained with dietary consumption, the mechanism of growth inhibition should be relevant to PCa, and genistein (a broad-spectrum in vitro protein-tyrosine kinase inhibitor) should have relatively specific kinase inhibitory effectsin vivo. These considerations were investigated by measuring growth inhibitory activity in a variety of PCa cell lines. Growth inhibitory effects were shown not to occur with concentrations below the low micromolar range (i.e., 3 logs above that attained in serum). In-depth mechanistic studies with the PC3-M metastatic variant cell line demonstrated that growth inhibition was independent of genistein’s estrogenic effects. Genistein was shown to decrease the viability of nonadherent cells, suggesting a lack of dependence on cell adhesion for growth inhibition. However, important molecular and kinetic differences between genistein’s effects on growth in adherent versus nonadherent cells were identified. Specific suppression of focal adhesion kinase activity (without global decreases in phosphotyrosine) was shown to precede induction of apoptosis, which was responsible for growth inhibition in adherent cells. These findings do not support anin vivo growth inhibitory role by genistein consumed in quantities associated with a soy-based diet. They do, however, identify genistein as a potential therapeutic agent for PCa and as a tool with which to study the control of apoptosis in PCa.

Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y.

Abstract: SUMMARY Chronic exposure to nicotine has been reported to increase the number of nicotinic acetylcholine receptors (AChRs) in brain. The mechanism of up-regulation for the a4b2 AChR subtype, which accounts for the majority of high affinity nicotine binding in mammalian brain, has previously been shown to involve a decrease in the rate of a4b2 AChR turnover. Here, we report an investigation of the extent and mechanism of nicotine-induced up-regulation of a3 AChRs and a7 AChR subtypes expressed in the human neuroblastoma cell line SH-SY5Y. Up-regulation of human a3 AChRs and a7 AChRs, unlike a4b2 AChRs, requires much higher nicotine concentrations than are encountered in smokers; the extent of increase of surface AChRs is much less; and the mechanisms of up-regulation are different than with a4b2 AChRs. The mechanisms of up-regulation may be different for a3 AChRs or a7 AChRs. Chronic treatment with nicotine or carbamylcholine, but not d-tubocurarine, mecamylamine, or dihydro-b-erythroidine, induced a 500 ‐ 600% increase in the number of a3 AChRs but only a 30% increase in a7 AChRs. Chronic nicotine treatment did not increase affinity for nicotine or increase the amount of RNA for a 3o r a 7 subunits. The effect of nicotine on up-regulation of a7 AChRs was partially blocked by either d-tubocurarine or mecamylamine. The effect of nicotine treatment on the number of a3 AChRs was only slightly blocked by the antagonists d-tubocurarine, mecamylamine, or dihydro-b-erythroidine at concentrations that efficiently block a3 AChR function. Most of the nicotineinduced increase in a3 AChRs was found to be intracellular. The a3 AChRs, which accumulate intracellularly, were shown to have been previously exposed on the cell surface by their susceptibility to antigenic modulation. The data suggest that chronic exposure to nicotine may induce a conformation of cell surface a3 AChRs that at least in this cell line are consequently internalized but not immediately destroyed.

Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

Abstract: The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

Subtype-Specific Intracellular Trafficking of α2-Adrenergic Receptors

Abstract: The three alpha2-adrenergic receptor subtypes (alpha2a, alpha2b, and alpha2c) are highly homologous G protein-coupled receptors. These receptors all couple to pertussis toxin-sensitive G proteins and have relatively similar pharmacological properties. To further explore functional differences between these receptors, we used immunocytochemical techniques to compare the ability of the three alpha2-receptor subtypes to undergo agonist-mediated internalization. The alpha2a-receptor does not internalize after agonist treatment. In contrast, we observed that the alpha2b-receptor is able to undergo agonist-induced internalization and seems to follow the same endosomal pathway used by the beta2-adrenergic receptor. Attempts to examine internalization of the alpha2c-receptor were complicated by the fact that the majority of the alpha2c receptor resides in the endoplasmic reticulum and cis/media Golgi and there is relatively little cell surface localization. Nevertheless, we were able to detect some internalization of the alpha2c-receptor after prolonged agonist treatment. However, we observed no significant movement of alpha2c-receptor from the intracellular pool to the plasma membrane during a 4-hr treatment of cells with cycloheximide, suggesting that these cells are unable to process alpha2c-receptors in the same way they process the alpha2a or alpha2b subtypes.