D1 and D2 dopamine receptors form heterooligomers and cointernalize after selective activation of either receptor

We provided evidence for the formation of a novel phospholipase C-mediated calcium signal arising from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity indicative of D1-D2 receptor heterooligomerization. The close proximity of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers.     

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

AIM: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. METHODS: Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. RESULTS: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor. CONCLUSION: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.     

Age related decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats

The densities (Bmax) and apparent dissociation constants (Kd) of D1 and D2 receptors in striatum and of 5-HT2 receptors in cortex of rats aged 1, 3, 7 and 12 months have been determined using 3H-SCH 23390, 3H-spiperone and 3H-ketanserin, respectively, as ligands. No changes in Kd's were seen. The density of D1 receptors decreases continuously from 1 month, attaining approximately 65% at 12 months of age. The density of D2 receptors increases slightly from 1 to 3 months, followed by a decrease until 12 months of age, where the level is approximately 80% of the 1 month value. The rise D2 receptor density is less apparent when calculated on a protein basis due to a concomitant increase in protein content of corpus striatum. The ratio between D1 and D2 receptors remains constant, approximately 4.2, from 1 to 12 months of age. The density of 5-HT2 receptors decreased from 1 to 7 months and remained at this level also after 12 months.     

3C疗法治疗1型糖尿病儿童维生素D水平研究

目的：评估维生素D与1型糖尿病（T1DM）患儿的临床关系,为T1DM的防治提供新的依据。方法：选取我院2017-2018年新诊断及使用3C疗法治疗的T1DM患儿,分析T1DM患儿与健康体检患儿血清25-羟维生素D［25(OH)D］水平。根据25(OH) D水平,将T1DM患儿分为3组（缺乏组、不足组及充足组）,比较三个亚组的性别、年龄、居住地、体质量指数（BMI）、空腹C肽、空腹血糖及胰岛素用量等情况,探讨不同tanner分期、性别、季节、有无合并糖尿病酮症酸中毒（DKA）与血清维生素D水平的关系。结果：T1DM组患儿血清25(OH)D水平为（42.31±22.01）nmol/L,较健康对照组的（50.37±22.28）nmol/L低,差异有统计学意义（P<0.05）。T1DM组患儿中,维生素D充足组的空腹C肽水平高于维生素D不足组及维生素D缺乏组（P<0.05）;维生素D缺乏组空腹血糖水平及单位体质量胰岛素用量高于维生素D充足组（P<0.05）。按血清25(OH)D水平测定时间不同分A组（1-3月、10-12月）和B组（4-9月）,A组25(OH)D水平低于B组（P<0.05）;合并DKA组25(OH)D水平低于无DKA组（P<0.05）。结论：T1DM患儿普遍存在维生素D缺乏,尤其是合并DKA、及1-3月及10-12月的患儿;维生素D充足的T1DM患儿可减少胰岛素用量。临床上要加强T1DM患儿的维生素D的监测以及补充。     

Selective inactivation of dopamine D1 and D2 receptors in 6-hydroxydopamine-lesioned rats: evidence that the effect of D1 and D2 agonists can be expressed in the absence of the heterologous DA receptor

EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) markedly decreased the density of dopamine (DA) D1 and D2 receptors in the lesioned and normal striatae of rats lesioned unilaterally with 6-hydroxy-DA. By treatment with either the D1 antagonist SCH 23390 or the D2 antagonist raclopride, together with EEDQ selective inactivation of D2 and D1 receptors, respectively, are obtained. In rats with decreased density of D1 receptors the circling behaviour response to the D1 agonist SK&F 38393 was markedly inhibited 24 hours after EEDQ treatment, whereas the similar response to the D2 agonist pergolide was unchanged. In rats with decreased density of D2 receptors the effects of pergolide and the partial D2 agonist (-)-3-PPP were antagonized, while the effect of SK&F 38393 was unchanged. These results indicate that the effect of D1 and D2 agonists can be expressed in the absence of normal densities of the heterologous DA receptor. In contrast, the responses from homologous DA receptors, mediating the circling behaviour from the denervated side of the brain, are highly sensitive to the inactivating effect of EEDQ.     

Spectrophotometric determination of vitamin A in oily capsules using first derivative curves

The first derivative curve (D1) of the absorption spectrum of vitamin A acetate in cyclohexane possesses a trough (D11) at 348 nm and a maximum (D12) at 306 nm. D11, D12, and delta D1 (= D11 - D12) are linearly related to concentration over a range of 5-25 i.u. ml-1. When a tangent is drawn between D1 at 400 nm and D1, at 306 nm, the amplitude at 348 nm (D1 corr.) is linearly related to concentration. Ratios of magnitude of D11/magnitude of D12 and D1(corr.)/magnitude of D1 are independent of concentration, and have been used to reveal the presence of interferences in the D1 curves of vitamin A in oily capsules. Vitamin A in two oily preparations has been assayed using delta D1 and D1(corr.). The potency found was within +/- 2% from that obtained by using the B.P. method. The method is rapid, precise and accurate.     

Comparative uptake, retention and cytotoxicity of daunorubicin by human myeloid cells

We studied the cellular uptake and retention of daunorubicin (D1) in two human leukemic cell lines (ML1 and K562) and myecloblasts from an untreated patient with acute myelogenous leukemia (AML). The rate of uptake and the steady-state level of D1 were not temperature dependent but increased markedly with increases in the pH of the medium. Also, saturation kinetics were not demonstrable using concentrations of D1 up to 111 microM. Together, these observation suggest a transport mechanism for D1 compatible with passive diffusion. Accumulation of D1 was increased only in cells from the AML patient with addition of sodium azide, whereas drug efflux was not increased significantly in the presence of glucose in MLI or K562 cells. Although the rate of uptake and steady-state levels of D1 were the same in these cells, metabolism and cytotoxicity of D1 differed. Our results indicate that ML1 cells can be used as a pharmacologic model for studying the metabolism and resistance of D1 in vivo.     

Functional interaction between dopamine D1 and D2 receptors in rat jaw movements.

The functional interaction between dopamine D1 and D2 receptors in dopamine-mediated jaw movements was studied in ketamine-anaesthetized rats after C1 spinal transection. Jaw movements were recorded by means of a light-emitting diode attached to the mandible; the method permits a detailed qualitative and quantitative analysis of jaw movements. D1 stimulation with SKF38393 (10 mg/kg i.v.) produced frequent bursts of teeth chattering, which were abolished by pretreatment with SCH23390 (0.25 mg/kg i.v.). D2 stimulation by quinpirole (1-10 mg/kg i.v.) produced infrequent bursts of jaw movements, which were characterized by low frequency jaw opening and closure movements from the rest position of the jaw, and absence of tongue protrusions. An additional stimulation of D1 receptors by giving SKF38393 30 min later produced an almost continuous pattern of jaw openings but less closure movements from the rest position, and the openings were accompanied by frequent tongue protrusions. These results clearly demonstrate that the type of oral behaviour produced by stimulation of D1 and D2 receptors together is qualitatively different from that produced by stimulation of either D1 or D2 receptors alone.     

人高密度脂蛋白受体CLA-1上调剂9179D的分离、结构鉴定和活性研究

目的从微生物代谢产物中分离能够上调人高密度脂蛋白受体（CLA-1）表达的新型活性化合物,并对其活性进行研究。方法应用已建立的CLA-1表达上调剂的模型,对阳性菌株链霉菌104A-9179发酵产物的活性成分进行分离纯化,获得活性化合物9179D;通过理化性质、质谱、紫外和核磁等波谱学数据进行结构鉴定;利用RT-PCR和Western Blot方法检测9179D对HepG2中CLA-1表达的影响;利用流式细胞仪检测其对小鼠巨噬细胞RAW264.7结合DiI-HDL的影响。结果从链霉菌104A-9179发酵产物中得到活性化合物9179D,并确定了其结构为曲占柳菌素D（Trichostatin D）;9179D在CLA-1上调剂模型上的EC₅₀为46.02μmol/L,表达活性最高值为934%;9179D能增加HepG2中CLA-1的mRNA和蛋白表达;增加RAW264.7对DiI-HDL的结合。结论得到一个微生物来源的具有强的上调CLA-1的表达活性的化合物-9179D（曲古柳菌素D）,属首次报道。     

Synthesis and dopamine receptor affinities of N-alkyl-11-hydroxy-2-methoxynoraporphines: N-alkyl substituents determine D1 versus D2 receptor selectivity

We developed a procedure to synthesize a series of N-alkyl-2-methoxy-11-hydroxynoraporphines from thebaine and evaluated their binding affinities at dopamine D1 and D2 receptors in rat forebrain tissue. At D2 receptors, the most potent 10,11-catechol-aporphine was (R)-(-)-2-methoxy-N-n-propylnorapomorphine (D2, Ki = 1.3 nM; D1, Ki = 6450 nM), and the most selective and potent 11-monohydroxy aporphine was (R)-(-)-2-methoxy-11-hydroxy-N-n-propylnoraporphine (D2, Ki = 44 nM; D1, Ki = 1690 nM). In contrast, the N-methyl congeners (R)-(-)-2-methoxy-11-hydroxy-N-methyl-aporphine (D1 vs D2, Ki = 46 vs 235 nM) showed higher D1 than D2 affinity, indicating that N-alkyl substituents have major effects on D2 affinity and D2/D1 selectivity in such 2-methoxy-11-monohydroxy-substituted aporphines.     

Reciprocal regulation of dopamine D1 and D3 receptor function and trafficking by heterodimerization

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET(2)), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET(2) signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves beta-arrestin binding because it was blocked by mutant beta-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.     

Cytotoxicity of some azines of acetophenone derived mono-Mannich bases against Jurkat cells

Acetophenone derived mono-Mannich bases (Ig1-Ig4), 1-aryl-3-amino-1-propanone hydrochlorides, which are known to have cytotoxicity in Jurkat cells, were synthesized. Then, they were converted to corresponding azine derivatives (D1-D4), N, N'-bis(3-amino-1-aryl-propylidene)hydrazine dihydrochlorides, which are bifunctional agents. The aryl part was replaced by phenyl in Ig1, Ig2, Ig3, D1, D2, and D3, and by p-hydroxyphenyl in Ig4 and D4. The amine part was replaced by dimethylamine in Ig1, D1, Ig4 and D4, by piperidine in Ig2 and D2, and by morpholine in Ig3 and D3. The aim of this study was to investigate whether the modification in chemical structure, converting the mono-Mannich base to a corresponding azine derivative, improves the cytotoxicity. In addition, the effect of the representative compound, D3, N, N'-bis(3-morpholine-4-yl-1-phenylpropylidene)hydrazine dihydrochloride, on cellular glutathione level after 1 h exposure in phosphate buffer at 37 degrees C was also determined to provide information on a possible mechanism of cytotoxic action. Compounds D2-D4 are reported for the first time in this study. Except for Ig2 and D2, the cytotoxicity of mono-Mannich bases, Ig1, Ig3 and Ig4 and corresponding azine derivatives, D1, D3 and D4 were higher than the reference compound 5-FU. Azine derivatives D1 and D4 had almost equal cytotoxic potency with corresponding mono-Mannich bases Ig1 and Ig4, respectively. On the other hand, azine derivatives D2 and D3, had 1.28 and 1.90-times less cytotoxicity in Jurkat cells compared with the mono-Mannich bases, Ig2 and Ig3, respectively, from which they are derived. Azine derivative D3 dose-dependently decreased the total cellular glutathione level, suggesting that azine derivatives may exert cytotoxicity by thiol alkylation. Azine derivatives with equal or less cytotoxic potency compared to the mono-Mannich bases they are derived from seemed to be less suitable derivatives for the development of new cytotoxic compounds.     

Parallel decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats from 3 to 25 months of age

The density (Bmax) and apparent dissociation constant (KD) of dopamine D1 and D2 receptors in striatum was estimated in rats of different ages (from 3.5 to 25 months) using 3H-SCH 23390 and 3H-spiperone as ligands. The density of D1 and D2 receptors decreases with age attaining 70 and 69% of the 3.5 months' value, respectively, whereas the KD's remain constant. The decreases in density of D1 and D2 receptors are parallel. Thus, throughout life the ratio between the density of D1 and D2 receptors remains constant.     

Alpha(1D)-adrenoceptors mediate nerve and agonist-evoked contractions in mouse vas deferens: evidence obtained from knockout technology

1 It has been demonstrated that nerve-evoked contractions of the rat vas deferens involve alpha(1D)-adrenoceptors. Definitive evidence for a similar alpha(1D)-adrenoceptor-mediated response in mouse vas deferens has been more difficult to obtain. In this study, we have used alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) mice to aid in the pharmacological characterization. 2 Mouse whole vas deferens was stimulated with a single pulse every 5 min. Once a stable response had been obtained, vehicle or antagonist was administered cumulatively at 5-min intervals and a response to stimulation obtained 5 min later. Cumulative concentration-response curves were also obtained for noradrenaline. 3 In vas deferens from alpha(1D)-KO mice, the contractile response to low concentrations of noradrenaline and the contractile response to a single stimulus were significantly reduced as compared to wild type (WT). 4 The alpha(1D)-adrenoceptor selective antagonist, BMY 7378, produced a concentration-dependent inhibition of single pulse-evoked contractions of vas deferens from WT and alpha(1D)-KO mice. BMY 7378 was significantly less potent in inhibiting stimulation-evoked contractions in vas deferens from alpha(1D)-KO mice. 5 It is concluded that alpha(1D)-adrenoceptors mediate a component of nerve- and agonist-evoked contractions of the vas deferens of WT mice.     

Regulation of dense core vesicle release from PC12 cells by interaction between the D2 dopamine receptor and calcium-dependent activator protein for secretion (CAPS)

We identified CAPS1 (calcium-dependent activator protein for secretion) as a D2 dopamine receptor interacting protein (DRIP) in a yeast two-hybrid screen of a human brain library using the second intracellular domain of the human D2 receptor (D2IC2). CAPS1 is an evolutionarily conserved calcium binding protein essential for late-stage exocytosis of neurotransmitters from synaptic terminals. CAPS1 interaction was confirmed for both the long and short isoforms of the D2 receptor, but not with any other dopamine receptor subtype. Interaction between CAPS1 and the D2 receptor was validated using both pulldown and coimmunoprecipitation assays. Deletion mapping localized the D2 receptor binding site to a segment located within the C-terminal region of CAPS1 as well CAPS2. In PC12 cells, CAPS1 and D2 receptors were found to colocalize within both cytosolic and plasma membrane compartments. Overexpression of a truncated D2 receptor fragment caused a significant decrease in K(+)-evoked dopamine release from PC12 cells, whereas no effect on norepinephrine or BDNF release was observed. These results suggest that D2 dopamine receptors may modulate vesicle release from neuroendocrine cells via direct interaction with components of the exocytotic machinery.     

知母中三个新的呋甾皂苷

从中药知母（ＡｎｅｍａｒｒｈｅｎａａｓｐｈｏｄｅｌｏｉｄｅｓＢｇｅ．）中分离出三种新的呋甾皂苷，初步鉴定为（２５Ｓ）－２６－Ｏ－β－Ｄ－葡萄糖－５β－呋甾－２０（２２）－双键－３β，２６－二醇－３－Ｏ－β－Ｄ－葡萄糖基（１→２）〔β－Ｄ－葡萄糖基（１→３）〕－β－Ｄ－葡萄糖基（１→４）－β－Ｄ－半乳糖苷（１），（２５Ｓ）－２６－Ｏ－β－Ｄ－葡萄糖基－２２－羟基－５β－呋甾－３β，２６－二醇－３－Ｏ－β－Ｄ－葡萄糖基（１→２）〔β－Ｄ－葡萄糖基（１→３）〕－β－Ｄ－葡萄糖基（１→４）－β－Ｄ－半乳糖苷（２），（２５Ｓ）－２６－Ｏ－β－Ｄ－葡萄糖基－２２－甲氧基－５β－呋甾－３β，２６－二醇－３－Ｏ－β－Ｄ－葡萄糖基（１→２）〔β－Ｄ－葡萄糖基（１→３）〕－β－Ｄ－葡萄糖基（１→４）－β－Ｄ－半乳糖苷（３）．分别命名为ｔｉｍｏｓａｐｏｎｉｎ－ＢⅣ，ｔｉｍｏ－ｓａｐｏｎｉｎ－ＢⅤ，ｔｉｍｏｓａｐｏｎｉｎ－ＢⅥ．     

Bioactive cyclic peptides from the psychrotolerant fungus Penicillium algidum

A new cyclic nitropeptide, psychrophilin D (1), together with two known cyclic peptides, cycloaspeptide A (2) and cycloaspeptide D (3), were isolated from the psychrotolerant fungus Penicillium algidum using C18 flash chromatography, LH-20 Sephadex and preparative HPLC. The structure of psychrophilin D (1) was derived from mass spectrometric information, 1D and 2D NMR spectra and Marfey's method. The compounds were tested in antimicrobial, antiviral, anticancer and antiplasmodial assays. Psychrophilin D (1) exhibited a moderate activity (ID50 10.1 microg/ml) in the P388 murine leukaemia cell assay. Cycloaspeptide A (2) and D (3) exhibited moderate activity (IC50 3.5 and 4.7 microg/ml, respectively) against Plasmodium falciparum.     

Depression linked to higher antibodies production against estrogenized insulin in type 1 diabetes

Depression has been commonly associated with type 1 diabetes (T1D) and insulin covalently modified with catecholestrogens (CEs) was found in serum of these T1D patients. This study aimed to know whether depression link to higher antibodies against estrogenized insulin in T1D.ELISA (direct binding and competition) and quantitative precipitin titration were used to detect antibodies and their affinities against estrogenized insulin in the serum of 66 depressed T1D (DT1D) patients (out of 110 T1D) and 41 control subjects. Antibodies from DT1D patients showed high binding specificity to estrogenized insulin (2-hydroestradiol-insulin; 2-OHE₂-Ins) in comparison to overall T1D patients (p < 0.05) or control subjects (p < 0.001). However, T1D sera demonstrate high recognition to 2-OHE₂-Ins as compared to Ins (p < 0.05) or 2-OHE₂ (p < 0.001). The affinity of antibodies from DT1D and T1D patients was 1.32 × 10^-7 M and 1.43 × 10^-7 M, respectively. Depression linked to higher antibodies production against estrogenized insulin in T1D. Furthermore, depression in T1D generates inflammatory conditions that further increased antibodies production in T1D patients.     

特异性结合白色念珠菌（1，3）-β-D-葡聚糖单链 DNA 适配子的筛选及其应用

目的：通过指数富集的配基系统进化技术（systematic evolution of ligands by exponential enrichment ,SELEX）筛选能与高亲和力（1,3）‐β‐D‐葡聚糖特异性结合的适配子,并用该适配子建立双适配子夹心酶联寡聚核苷酸吸附试验（enzyme‐linked oligonucleotide assay ,ELONA）来对深部真菌感染血浆进行辅助诊断。方法提取白色念珠菌 ATCC10231株细胞壁（1,3）‐β‐D‐葡聚糖,通过 SELEX 筛选获得能与（1,3）‐β‐D‐葡聚糖特异性结合的高亲和力单链 DNA 适配子,并用该适配子建立双适配子夹心ELONA 来检测深部真菌感染血浆中的（1,3）‐β‐D‐葡聚糖。结果从白色念珠菌细胞壁中成功提取了高聚合度（1,3）‐β‐D‐葡聚糖,并通过体外酶解获得可溶性低聚合度（1,3）‐β‐D‐葡聚糖作为筛选靶标。用SELEX 进行12轮筛选后,从初始单链 DNA 文库中获得2个高亲和力适配子 AU1和 AD1,检测发现它们并非结合于（1,3）‐β‐D‐葡聚糖的同一表位。双适配子夹心 ELONA 检测深部真菌感染血浆中（1,3）‐β‐D‐葡聚糖的特异性和敏感度分别为91．94％和92．31％。结论从初始单链 DNA 文库中成功获得可与（1,3）‐β‐D‐葡聚糖特异结合的高亲和力适配子,为深部真菌感染新型诊断试剂的研发奠定了基础。