Organic Anion and Cation Transport in Vitro by Dog Choroid Plexus: Effects of Neuroleptics and Tricyclic Antidepressants

Abstract Dog lateral choroid plexus accumulates the cation ¹⁴C-emepronium and the divalent anion ¹²⁵I-iodipamide in vitro. At 10 μM, high potency neuroleptics with a substituted piperazine side chain and also haloperidol depress only the uptake of the cation and even stimulate the uptake of the anion. In contrast, at 1-10 μM, the accumulation of both test substances is inhibited by neuroleptics and tricyclic antidepressants with an aliphatic side chain. Such unspecific effects on seemingly unrelated transport systems at concentrations reached clinically in the CSF might explain some side actions of low potency neuroleptics and antidepressants.     

Differences between Binding Affinities of some Antimuscarinic Drugs in the Parotid Gland and those in the Urinary Bladder and Ileum

Abstract: Possible differences between the muscarinic receptors in the guinea pig urinary bladder and those in the ileum and the parotid gland were investigated, using a receptor binding technique. The affinities of 18 antimuscarinic drugs were indirectly derived from the ability to inhibit the receptor-specific binding of the radioligand (–)³H-QNB. The Hill coefficients were close to unity which indicated that the drugs were bound to apparently uniform populations of receptors within each tissue. In contrast to traditional muscarinic antagonists, four drugs – namely, oxybutynine, dicyclomine, benzhexol and pirenzepine – bound with a significantly higher affinity in the parotid gland than in the urinary bladder and ileum. A tendency towards reversed selectivity was found for secoverine. Thus, the present results further support the hypothesis that differences in muscarinic receptors between tissues exist, e.g. smooth muscle compared with parotid gland, which can be detected only by certain antimuscarinic drugs.     

The Influence of Lithium on Water Binding Ability,Consistency and Macromolecules in the Rat Brain

Abstract Wistar male rats were treated with lithium for 50, 80, and 120 days, respectively. Lithium was added to the diet (40 mmol/kg of diet), the plasma lithium concentration being in the range of 0.55–0.70 mmol/l of plasma. The treatment caused a significantly more rigid consistency of the brain tissue. The water content and water binding ability in various parts of the brain - measured by a drying course - was unchanged. The content of hexosamine and protein in total brains was significantly decreased (about 10 per cent) after the treatment. The content of chondroitin sulphate in various parts of the brain was unchanged, whereas the content of hyaluronic acid was significantly increased in the cerebellum (25 per cent) and in the thalamic area (20 per cent). The results suggest an influence of lithium on the macromolecules from the extracellular space, causing an alteration of the neuronal function.     

Stereospecific Distribution of Methylphenidate Enantiomers in Rat Brain: Specific Binding to Dopamine Reuptake Sites

To investigate the stereoselective distribution of methylphenidate (MPD) enantiomers in rats, the concentrations of each enantiomer were determined in plasma and brain regions (cerebellum, striatum, basal forebrain, brain stem, and cortex) after iv administration of racemic MPD and its individual enantiomers. The concentrations of MPD enantiomers in each brain region reached pseudo-steady state within 10 min after iv administration of racemic MPD (2 mg/kg dose). The influx clearances for MPD calculated from K _Papp values in each brain region were not significantly different between MPD enantiomers and between the five brain regions. The mean K _Papp values for (+ )-MPD in the striatum at 120 and 240 min after administration of racemic MPD were 10.1 and 10.5, respectively, and these values at each time were significantly larger than the K _Papp values (7.5 and 7.0, respectively) for the (–)-isomer (P < 0.01). The K _Papp value for (+ )-MPD in the striatum decreased by coadministration of mazindol as an inhibition of both dopamine and norepinephrine reuptake, but it was not changed by desipramine as a norepinephrine reuptake inhibitor. These results suggest that ( + )-MPD was bound specifically to the dopamine reuptake site in the striatum.     

Structural and Pharmacological Differences between Codfish and Rat Brain α1-Adrenergic Receptors Revealed by Photoaffinity Labeling with 125I-APDQ

Abstract: The photoaffinity probe ¹²⁵I-APDQ has been used to characterize α₁-receptor peptides in the cod and rat brains. In the cod brain a major specific peptide of M_t = 68,000 could be covalently labeled by ¹²⁵I-APDQ as revealed by SDS-PAGE. In the rat brain a specific peptide with M_t = 77,000 was instead labeled. When a number of adrenergic agonists and antagonists were tested for their ability to protect the labeling by ¹²⁵I-APDQ their potencies were those expected for α₁-receptors in both species. The ligand binding peptide in the cod brain also distinguished between stereoisomers of epinephrine as expected for a physiological receptor. However, there was a distinct difference between the cod and rat α₁-receptor in that the β-agonist 1-isoprenaline was equipotent to 1-norepinephrine in the cod whereas it was less potent in the rat. The protecting ability of the tested agents were also matched by their ability to displace the α₁-adrenergic ligand ³H-prazosin from α₁-receptor binding sites in brain membranes from both species. Thus, the codfish α₁-receptor seems to be different from mammalian α₁-receptors both structurally and pharmacologically.     

基于主成分分析-聚类分析法的不同商品规格白芍饮片中5种成分比较及质量评价研究

目的 建立白芍中5种主要化学成分的HPLC测定方法,综合评价不同等级白芍饮片的质量,为白芍饮片的商品规格等级标准研究提供参考。方法 采用HPLC,SunFire^® C₁₈（4.6 mm×250 mm,5μm）色谱柱,流动相乙腈（A）-0.05%磷酸水溶液（B）梯度洗脱。建立外标法同时测定没食子酸、芍药内酯苷、芍药苷、1,2,3,4,6-五没食子酰葡萄糖（1,2,3,4,6-penta-O-galloyl-β-D-glucose,β-PGG）和苯甲酰芍药苷5种成分含量;采用主成分分析-聚类分析法的模式进行多元统计学分析。结果 一等、二等、三等、统货及鲜切饮片5种规格等级的样品中5种成分平均含量分别为没食子酸1.000 9,1.016 4,0.949 6,1.039 7,0.974 7 mg·g^-1,芍药内酯苷5.000 6,5.336 6,6.124 0,5.449 8,9.922 2 mg·g^-1,芍药苷30.212 9,29.478 3,30.186 7,29.516 6,35.251 1 mg·g^-1,β-PGG 3.250 3,2.498 1,3.704 9,3.736 9,3.710 0 mg·g^-1和苯甲酰芍药苷0.651 8,0.754 7,0.782 0,0.711 3,0.748 9 mg·g^-1。主成分分析结果表明,以鲜切饮片相对较好,其他等级饮片与饮片直径大小未体现较强的正相关性;聚类分析结果表明,判别距离<5时,分为5个大类,11批鲜切饮片单独一类,其他等级交互成4类。结论 该含量测定方法重复性、仪器精密度、回收率表现良好;以该5种成分的含量为指标,基于多元统计分析评价不同等级白芍饮片质量,为白芍饮片的商品规格等级标准研究提供参考。     

Lectin-Mediated Drug Targeting: Selection of Valency, Sugar Type (Gal/Lac), and Spacer Length for Cluster Glycosides as Parameters to Distinguish Ligand Binding to C-Type Asialoglycoprotein Receptors and Galectins

Purpose. Common oligosaccharides of cellularglycoconjugates are ligands for more than one type of endogenous lectin.Overlapping specificities to -galactosides of C-type lectins andgalectins can reduce target selectivity of carbohydrate-ligand-dependentdrug targeting. The purpose of this study is to explore distinct features ofligand presentation and structure for design of cluster glycosides todistinguish between asialoglycoprotein-specific (C-type) lectins andgalectins. Methods. Extent of binding of labeled sugar receptors totwo types of matrix-immobilized (neo)glycoproteins and to cells wasevaluated in the absence and presence of competitive inhibitors. This panelcomprised synthetic mono-, bi-, and trivalent glycosides with two spacerlengths and galactose or lactose as ligand part. Results. In contrast to C-type lectins of hepatocytes andmacrophages, bi- and trivalent glycosides do not yield a notable glycosidecluster effect for galectins-1 and -3. Also, theseCa²⁺-independent galactoside-binding proteins prefer to homein on lactose-bearing glycosides relative to galactose as ligand, whilespacer length requirements were rather similar. Conclusions. Trivalent cluster glycosides with Gal/GalNAcas ligand markedly distinguish between C-type lectins and galectins.Undesired side reactivities to galectins for C-type lectin drug deliverywill thus be minimal.     

Comparison of the Methods for Determining Cell-Surface and Intracellular Receptors for Epidermal Growth Factor in the Rat Liver

We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37°C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intra-cellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 µg/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.     

A Comparison of the Binding Characteristics of the β-Adrenoceptor Antagonists 3H-Dihydroalprenolol and 125I-Iodocyanopindolol in Rat Liver

Abstract: The binding characteristics of ³H-dihydroalprenolol and ¹²⁵I-iodocyanopindolol have been compared in a particulate fraction from regenerating rat liver. When total ³H-dihydroalprenolol binding and inhibition of total ³H-dihydroalprenolol binding by (-)isoprenaline, (-)alprenolol and (±)cyanopindolol was investigated, it was found that all agents were bound to two classes of saturable binding sites. In the inhibition studies, the presence of two binding components was not obvious until the data were transformed into Hofstee plots and these were decomposed, except in the case of (±)cyanopindolol. Only (±)cyanopindolol was found to distinguish clearly between the two saturable binding sites identified by ³H-dihydroalprenolol, as indicated by a broad plateau in the inhibition curve. When ¹²⁵I-iodocyanopindolol was used as radioligand, only one saturable binding site was identified, even in the presence of less selective inhibiting ligands. The lower affinity component of ³H-dihydroalprenolol binding could be inhibited by 10 μM phentolamine. However, binding experiments with ³H-prazosin indicated that the lower affinity component was not identical with the alpha-adrenoceptor. Phentolamine did not influence ¹²⁵I-iodocyanopindolol binding. Thus, due to its higher specific activity and a high degree of selectivity, ¹²⁵I-iodocyanopindolol appears to be the ligand of choice.     

Activity of 2,3-benzodiazepines at Native Rat and Recombinant Human Glutamate Receptors In Vitro: Stereospecificity and Selectivity Profiles

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYK152466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 μM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate ₅₀ values of 18 μM, 24 μM and 6 μM, respectively and AMPA (10 μM) responses in recombinant human GluR4 expressing HEK293 cells with approximate ₅₀ values of 22 μM, 28 μM and 5 μM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate ₅₀ values of 10 μM, 8 μM and 1.5 μM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (−) isomer of each compound. At a concentration of 100 μM, GYKI52466, LY293606 and LY300168 produced <30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 μM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 μM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate ₅₀ value of 1 μM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor. © 1997 Elsevier Science Ltd. All rights reserved.     

Assay of Adenylate Cyclase in Rat Brain Homogenates and Lysed Turkey Erythrocytes: Two Different Methods give Different Results with Brain but not with Erythrocytes

Abstract: Experiments showed that the cyclic AMP (cAMP) fraction isolated by alumina or Dowex 50/ alumina chromatography from brain adenylate cyclase reaction mixtures contained “P radioactivity 10–12 times in excess of that which could be accounted for by determination of cAMP using binding assays. No such discrepancy was found when lysed turkey erythrocytes were assayed. This indicated that special precautions must be taken for the purification of ³²PcAMP from brain adenylate cyclase assays due to the formation of ³²P-labelled contaminants.     

Pharmacological Properties of Cannabinoid Receptors in the Avian Brain: Similarity of Rat and Chicken Cannabinoid1 Receptor Recognition Sites and Expression of Cannabinoid2 Receptor‐Like Immunoreactivity in the Embryonic Chick Brain

Abstract: The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day‐old chickens, since little is known about the avian cannabinoid system. The cannabinoid₁ receptor‐selective antagonist ligand [³H]SR 141716A bound to chicken brain membranes with K_D and B_max values of 0.92±0.28 nM and 790±58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI₅₀ value of 7.63±0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212–2>R‐1 methanandamide≈DAK. S(?)WIN 55,212–3 and AM404 were without inhibitory effect at 1 μM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non‐hydrolysable GTP analogues Gpp[NH]p and GTPγS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G‐proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [³H]SR 141716A to chicken membranes with a pI₅₀ value of 6.39±0.16. Using a novel antibody raised to amino acids 346–359 from the C‐terminal tail of the human cannabinoid₂ receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a ～53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid₂ receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a “classical” cannabinoid₂‐receptor component of [³H]WIN 55212‐2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid₂‐receptor‐selective antagonist SR 144528) was not found.     

Discovering the Hidden Profiles of Employee Drinking Motives: An Application of Integrated Variable- and Person-Centered Latent Profile Analysis

The drinking motives questionnaire (DMQ, Cooper, 1994) has been a very useful measurement tool for understanding why people drink alcohol. Recent attempts to examine drinking motives used the DMQ within a person-centered analysis framework. However, latent profiles identified in previous research largely presented level effects without strong shape effects, which consequently restricted meaningful interpretations and effective applications of drinking-motive profiles. To address this limitation, we applied a new alternative methodology for the study of drinking motives that integrated variable- and person-centered approaches. Our research clearly demonstrated that controlling for an overarching general drinking-motive construct provided a clearer disaggregation of shape and level effects. Four latent profiles were identified that represented a combination of shape and level effects. Each profile predicted different patterns of alcohol use. Theoretical as well as practical implications are discussed.     

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor,with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHR_HB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHR_HB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8^D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8^D6A showed 50% lower IC₅₀ compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8^D6A interfered with the binding of FSH to its receptor.     

Methotrexate Concentrations in Biological Fluids: Comparison of Results Obtained by Radioimmunoassay and Direct Ligand Binding Radioassay

Abstract A sensitive (sensitivity 2.2 × 10^-9 mol/1) and specific (practically no cross-reaction with circulating folates) radioimmunoassay for the determination of methotrexate concentrations in biological fluids is described and compared with a commercial competitive protein binding assay. Antiserum with high titer was produced in rabbits immunized with MTX-human serum albumin conjugate. Fitness for use in pharmacokinetic drug level determinations was shown in three patients, who received both low doses and high dose therapy combined with citrovorum factor rescue. An excellent correlation was found between plasma and urine MTX concentrations obtained by RIA and competitive protein binding assay. A two-compartment pharmacokinetic model was found adequately describing the serum decay curves, but there was a great interindividual variability in the calculated pharmacokinetic parameters.     

不同产地板蓝根中重金属元素的含量测定及分析

目的 测定25批不同产地的板蓝根药材中铅、镉、砷、汞、铜的含量。方法 通过微波消解-电感耦合等离子质谱法测定25批板蓝根药材中铅、镉、砷、汞和铜的含量,并运用SPSS 16.0软件对数据进行聚类分析。结果 25批板蓝根药材中,镉、砷、汞均有不同程度的超出规定限度,铅和铜均符合现行标准。结论 该方法分析了不同产地板蓝根中重金属含量差异特点及超标的可能原因,为板蓝根药材的规范化种植,安全评价及重金属含量标准制定提供一定依据。     

抗感冒药物成分的药理特征及其临床用药分析

目的:分析抗感冒药成分的药理特征及合理用药。方法:采用临床病例讨论,分析抗感冒药的合理使用。结果:抗感冒药所含成分类同,临床应用存在诸多问题。结论:了解患者所患感冒病症,询问病情,合理使用,减少或避免药物不良反应发生。     

不同产地茯苓中7种三萜类成分含量的测定及聚类分析

目的:建立同时测定茯苓药材中7种三萜类成分含量的方法,并比较不同产地茯苓药材中上述成分的差异性,为茯苓药材的质量控制提供参考。方法:以不同产地的36批茯苓药材为样品,采用高效液相色谱法测定去氢土莫酸、猪苓酸C、3-表去氢土莫酸、3-O-乙酰基-16α-羟基-氢化松苓酸、去氢茯苓酸、茯苓酸、松苓新酸的含量。色谱柱为Thermo Acclaim 120 C₁₈,流动相为乙腈-磷酸水(梯度洗脱),流速为1 mL/min,检测波长为210 nm,柱温为30℃,进样量为20μL。采用SPSS 21.0统计学软件对36批不同产地茯苓药材进行聚类分析。结果:7种三萜类成分在各自质量浓度范围内线性关系均良好(r均不低于0.999 0),平均加样回收率为96.74%～104.04%(RSD为0.54%～1.55%,n=6);精密度、重复性、稳定性(24 h)试验的RSD均小于3.0%(n=6);耐用性试验的RSD均小于5.0%(n=2)。不同产地样品之间7个指标成分的单一含量均存在一定差异,但总体差异不明显(多数样品总含量分布在1.3～1.9 mg/g之间)。经聚类分析,36批样品...     

扩张型心肌病大鼠心脏左室内皮素受体及其亚型分析

目的 观察扩张型心肌病大鼠左室ETR及其亚型的变化.方法用125I-ET-1建立内皮素受体及其亚型的放射配基分析法,进行饱和结合试验,对扩张型心肌病大鼠左室心肌ETR及其亚型的含量进行分析.结果(1) 放射配基分析法检测ETR的基本实验条件为37 ℃孵育 60 min,膜蛋白投放量以20～40 μg 为佳; (2) ET-1及非选择性拮抗剂bosentan、选择性拮抗剂BQ123、BQ788等均能竞争抑制125I-ET-1与内皮素受体的结合,而去甲肾上腺素则不能抑制; (3) 正常大鼠左室内皮素受体数量为 (92.21±34.34) nmol·     

Mutational analysis of the human complement 5a receptor: Identification of a potential role of asp 37 and asp 82 in ligand binding

The receptor for the inflammatory and chemotactic agent complement 5a (C5a) is a member of the G-protein coupled receptor (GPCR) superfamily. Site-directed mutagenesis of the human C5a receptor was performed to determine which amino acids were important for ligand binding. Specific regions of the C5a receptor were mutated based on their similarities to the ligand binding domain of other GPCRs. These mutated receptors were then transiently expressed in COS-7 cells in order to test their ability to bind [¹²⁵|]C5a. Because of the basic nature of the ligand, we concentrated on mutating acidic amino acid residues located at the N-terminal and transmembrane regions of the receptor. Mutation of Asp 37, located near the first transmembrane domain, or Asp 82, located within the second transmembrane domain, to valine resulted in a total loss of specific [¹²⁵l]C5a binding to membrane preparations of transfected cells. Furthermore, mutation of Asp 82 to alanine, leucine, or glutamate also resulted in an absence of specific binding. However, mutation of Asp 82 to asparagine did not eliminate the ability of the receptor to bind [¹²⁵l]C5a. Mutation of each of the N-terminal extracellular domain aspartate residues, Asp 282 (located within the seventh transmembrane domain), or Glu 179 or Glu 180 (located within the second extracellular loop) to valine also did not significantly affect [¹²⁵l]C5a binding. These studies thus identified two acidic amino acid residues of the C5a receptor which are important for binding [¹²⁵l]C5a. © 1995 Wiley-Liss, Inc.