Dopamine D1 receptor activation induces dehydroepiandrosterone sulfotransferase （SULT2A1） in HepG2 cells

Aim： Dopamine receptors are present in the nervous system and also widely distributed in the periphery. The aim of this study was to investigate the role of D1 subtype dopamine receptors （DRD1） in the regulation of dehydroepiandrosterone sulfotransferase （SULT2A1） in HepG2 cells. Methods： HepG2 cells were treated with DRD1 agonists with or without DRD1 antagonist for 9 d. DRD1 and SULT2A1 mRNA expression, protein expression, and SULT2A1 activity were detected using RT-PCR, Western blotting and HPLC, respectively. The level of cAMP was measured using a commercial kit. Results： All the 5 DR subtypes （DRD1-DRD1） were found to be expressed in HepG2 cells. Treatment of HepG2 cells with the specific DRD1 agonists SKF82958 （2.5 μmol/L） or SKF38393 （5 and 50 μmol/L） significantly increased the mRNA and protein expression of both DRD1 and SULT2A1, and increased SULT2A1 activity and cAMP levels. These effects were partially blocked by co-treatment with the specific DRD1 antagonist SCH23390 （2.5 μmol/L）. In addition, transfection of HepG2 cells with DRD1-specific siRNAs decreased DRD1 mRNA expression by 40%, which resulted in the reduction of SULT2A1 mRNA expression by 60%, protein expression by 40%, and enzyme activity by 20%. Conclusion： DRD1 activation upregulates DRD1 and SULT2A1 expression and SULT2A1 activity in HepG2 cells, suggesting that the DRD1 subtype may be involved in the metabolism of drugs and xenobiotics through regulating SULT2A1.     

Differential regulation of human α1-adrenoceptor subtypes

We have compared the agonist-induced downregulation of human α_1A-, α_1B- and α_1D-adrenoceptors upon stable expression in rat-1 fibroblasts. During a 24-h incubation the agonist phenylephrine downregulated α_1A- and α_1B-adrenoceptors in a concentration-dependent manner. While maximum downregulation was similar for both subtypes, the threshold concentration for significant reductions was markedly higher for α_1A- than for α_1B-adrenoceptors (10 μM vs. 100 nM). The downregulation of both subtypes by 100 μM phenylephrine was time-dependent, and significant reductions were observed already after 2– 4 h. In contrast, incubation of α_1D-adrenoceptor-expressing cells with phenylephrine increased receptor number in a time- and concentration-dependent manner. The downregulation of α_1B-adrenoceptors by 100 μM phenylephrine for 24 h was accompanied by a matching reduction in mRNA abundance, but no such reduction was seen for α_1A-adrenoceptors. These treatment conditions also caused a functional desensitization of agonist-stimulated inositol phosphate formation for α_1A- and α_1B- but not for α_1D-adrenoceptors. Treatment with the phorbol ester phorbol-12-myristate-13-acetate did not change receptor density or mRNA abundance and did not cause functional desensitization. We conclude that human α₁-adrenoceptor subtypes are differentially regulated by agonist treatment even if they are expressed in the same cell line. Received: 17 April 1998 / Accepted: 26 March 1999     

Effects of anthocyanins on the AhR–CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food–drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)–cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 μM concentration. PEL-2 and CYA-3 at 100 μM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR–CYP1A1 signaling, implying zero potential of these compounds for food–drug interactions with respect to AhR–CYP1A1 pathway.     

MiR-106b对子宫肌瘤细胞增殖、侵袭和凋亡的影响及与SULT2A1的靶向关系

摘要：目的:探讨微小RNA（miR）-106b在子宫肌瘤细胞增殖、侵袭和凋亡中的作用与潜在机制。方法:将体外培养的人子宫肌瘤细胞分为阴性对照（NC）组（转染miR-106b模拟物阴性对照）、miR-106b组（转染miR-106b模拟物）、反义（anti）-NC组（转染miR-106b抑制剂阴性对照）和anti-miR-106b组（转染miR-106b抑制剂）,采用逆转录-聚合酶链反应（RT-PCR）检测各组细胞中miR-106b的表达,噻唑蓝（MTT）法检测细胞存活率,平板克隆实验检测细胞的克隆形成能力,Transwell小室检测细胞侵袭,流式细胞仪检测细胞凋亡;生物信息学软件预测、双荧光素酶报告基因实验检测miR-106b和磺基转移酶2A1（SULT2A1）的靶向关系,免疫印迹（Western blotting）法检测miR-106b对SULT2A1蛋白的调控作用。结果:与NC组比较,miR-106b组细胞中miR-106b的表达水平、细胞存活率、克隆形成率、侵袭细胞数均明显升高,细胞凋亡率和SULT2A1蛋白的表达水平均明显降低（P<0.05）;与anti-NC组比较,anti-miR-106b组细胞中miR-106b的表达水平、细胞存活率、克隆形成率、侵袭细胞数均明显降低,细胞凋亡率和SULT2A1蛋白的表达水平均明显升高（P<0.05）。生物信息学软件Target Scan预测到miR-106b与SULT2A1 3’UTR存在互补的结合位点;双荧光素酶报告基因实验证实SULT2A1是miR-106b的靶基因。结论:miR-106b可促进子宫肌瘤细胞增殖、侵袭并抑制其凋亡,其作用机制可能与靶向调控SULT2A1表达有关。     

An expressional system of human cytochrome P-450 CYP1A1 gene transcription

目的：在人ＨｅｐＧ２细胞系上，建立人细胞色素Ｐ４５０ＣＹＰ１Ａ１（ＣＹＰ１Ａ１）基因转录的表达系统．方法：瞬时转染含人ＣＹＰ１Ａ１启动子的质粒（ｐＭＣ６３Ｋ）、ＥＬＩＳＡ法测定报道基因氯霉素乙酰转移酶（ＣＡＴ）的含量和酶学测定ＣＹＰ１Ａ１活性．结果：β萘黄酮２．５μｍｏｌ·Ｌ－１明显增强ＣＡＴ表达和ＣＹＰ１Ａ１活性（Ｐ＜０．０１）；在２．５－１０μｍｏｌ·Ｌ－１范围内，ＣＡＴ表达随浓度增高而不断增强，而ＣＹＰ１Ａ１活性则接近最高水平；１０μｍｏｌ·Ｌ－１时它们的作用强度分别为对照组的９４．３和２．８倍．用这种方法对八种含不同侧链的芥子油苷进行检测的结果表明，芸苔苷的水解产物（而非吲哚３原醇）诱导ＣＹＰ１Ａ１基因表达．结论：ＣＹＰ１Ａ１基因转录表达系统具有较高的可靠性和灵敏度     

Functional α1-adrenergic receptor subtypes in human right gastroepiploic artery

AIM: To study the functional alpha1-adrenergic receptor (alpha1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of alpha2-AR, alpha1-AR, and alpha1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by alpha2-AR selective antagonist yohimbine (pA2 6.82+/-0.28, slope 1.12+/-0.40),alpha1-AR selective antagonist prazosin (pA2 9.77+/-0.22, slope 0.90+/-0.22),alpha1A-AR selective antagonists RS17053 (pA2 8.42+/-0.20, slope 0.93+/-0.20) and 5-MU (pA2 8.42+/-0.22, slope 0.88+/-0.18),alpha1D-AR selective antagonist BMY7378 (pA2 6.84+/-0.32, slope 1.05+/-0.17), and alpha1A-,alpha1B-AR selective antagonist WB4101 (pA2 8.88+/-0.20, slope 1.15+/-0.16). The correlation coefficients between these pA2 values of alpha1-AR selective antagonists with pKi values of which obtained from alpha1A-, alpha1B- and alpha1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an alpha1B- and alpha1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9+/-0.5 to 5.6+/-0.6 and the maximal contraction was changed from (8.9+/-3.2) g to (8.0+/-3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by alpha1-AR, of which alpha1A-AR plays an important role, whereas alpha1B- and alpha1D-AR are not involved in the contraction response.     

Modulation of the human ρ1 GABAA receptor by inhibitory steroids

Rationale

Modulators of the ρ1 GABA_A receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood.

Objectives

We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABA_A receptor.

Results

Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3α,5β)-3-hydroxypregnan-20-one (3α5βP) while the T298F mutation eliminated inhibition by 17β-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3α5βP but was without effect on modulation by (3α,5β)-3-hydroxypregnan-20-one sulfate or 17β-estradiol. The effect of 3α5βP on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5β-reduced steroids containing an acetyl group on C17.

Conclusions

The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ρ1 GABA_A receptor: 5β-reduced uncharged steroids, sulfated and carboxylated steroids, and 17β-estradiol. Further division among 5β-reduced uncharged steroids was based on substituent at position C17.     

Binding ofβ-adrenoceptor blocking drugs to human serum albumin,to α1-acid glycoprotein and to human serum

Summary The binding of 8 -adrenergic blocking drugs to human serum albumin, to ₁-acid glycoprotein and to serum from normal volunteers and from patients with rheumatoid arthritis was studied. Protein binding was determined in vitro using equilibrium dialysis of labelled drug at 25° C. Oxprenolol and propranolol were highly bound to serum, alprenolol, pindolol and timolol to a lesser degree, and atenolol, metoprolol and sotalol were negligibly bound. For the five compounds which were appreciably bound, the mean binding was significantly higher in serum from patients with rheumatoid arthritis than in serum from normal volunteers. For those drugs, binding to ₁-acid glycoprotein was higher than to human serum albumin, and binding to a mixture of both proteins approached that to serum from healthy volunteers. For each of these drugs there was a strong correlation between the serum ₁-glycoprotein concentration and the percentage binding.     

Metabolic activation of 1 α-hydroxyvitamin D 3 in human liver microsomes

1. 1α -Hydroxyvitamin D 3 (1 α-OH-D 3) is a synthetic prodrug of the active form of vitamin D 3, and requires the hydroxylation at the C-25 position before eliciting its biological activity. 2. 25-Hydroxylation activities for 1 α-OH-D 3 were present in both microsomal and mitochondrial fractions of human liver. 3. To determine the P450 enzyme(s) involved in microsomal 25-hydroxylation, 14 P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2D6- Met, 2E1, 3A4, 4A11) were tested for their 25-hydroxylation activity of 1 α-OH-D 3. None catalysed the 25-hydroxylation reaction. 4. 1 α-OH-D 3 in a high concentration (2.5 ng ml -1) showed small but significant inhibition of the catalytic activities of CYP2C8, 2C9-Cys, 2C19, 2D6-Val and 2E1 for their typical substrates. However, 1 α-OH-D 3 in a clinically used low concentration will not significantly affect drug metabolism catalysed by the 14 P450s tested. 5. In summary, the 25-hydroxylation activity of 1 α-OH-D 3 that localizes in the microsomal fraction appears to be attributable to a cytochrome P450 other than the microsomal forms tested in this study.     

Effect of endothelin 1 on the isolated human and rabbit platelet activation

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High‐density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin‐1

Background and Purpose

Monocyte‐derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High‐density lipoproteins (HDLs) have many cardio‐protective properties including potent anti‐inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur.

Experimental Approach

Human blood monocytes were differentiated into macrophages in the presence or absence of HDL and were then induced to either an inflammatory macrophage (M1) or anti‐inflammatory macrophage (M2) phenotype using LPS and IFN‐γ or IL‐4, respectively.

Key Results

HDL inhibited the induction of macrophages to an M1‐phenotype, as evidenced by a decrease in the expression of M1‐specific cell surface markers CD192 and CD64, as well as M1‐associated inflammatory genes TNF‐α, IL‐6 and MCP‐1 (CCL2). HDL also inhibited M1 function by reducing the production of ROS. In contrast, HDL had no effect on macrophage induction to the M2‐phenotype. Similarly, methyl‐β‐cyclodextrin, a non‐specific cholesterol acceptor also suppressed the induction of M1 suggesting that cholesterol efflux is important in this process. Furthermore, HDL decreased membrane caveolin‐1 in M1 macrophages. We confirmed that caveolin‐1 is required for HDL to inhibit M1 induction as bone marrow‐derived macrophages from caveolin‐1 knockout mice continued to polarize into M1‐phenotype despite the presence of HDL. Moreover, HDL decreased ERK1/2 and STAT3 phosphorylation in M1 macrophages.

Conclusions and Implications

We concluded that HDL reduces the induction of macrophages to the inflammatory M1‐phenotype via redistribution of caveolin‐1, preventing the activation of ERK1/2 and STAT3.

Linked Articles

This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2016.173.issue-4

Abbreviations

BMDMs

bone marrow‐derived macrophages

Cav‐1^−/−

caveolin‐1 knockout

HDL

high‐density lipoprotein

MβCD

methyl‐beta‐cyclodextrin

M1

inflammatory macrophage

M2

anti‐inflammatory macrophage

WT

wild‐type

     

Inhibition behavior of fructus psoraleae’s ingredients towards human carboxylesterase 1 (hCES1)

1.?Fructus psoraleae (FP) is the dried ripe seeds of Psoralea corylifolia L. (Fabaceae) widely used in Asia, and has been reported to exert important biochemical and pharmacological activities. The adverse effects of FP remain unclear. The present study aims to determine the inhibition of human carboxylesterase 1 (CES1) by FP’s major ingredients, including neobavaisoflavone, corylifolinin, coryfolin, psoralidin, corylin and bavachinin.

2.?The probe substrate of CES1 2-(2-benzoyl-3-methoxyphenyl) benzothiazole (BMBT) was derived from 2-(2-hydroxy-3-methoxyphenyl) benzothiazole (HMBT), and human liver microsomes (HLMs)-catalyzed BMBT metabolism was used to phenotype the activity of CES1. In silico docking method was employed to explain the inhibition mechanism.

3.?All the tested compounds exerted strong inhibition towards the activity of CES1 in a concentration-dependent behavior. Furthermore, the inhibition kinetics was determined for the inhibition of neobavaisoflavone, corylifolinin, coryfolin, corylin and bavachinin towards CES1. Both Dixon and Lineweaver–Burk plots showed that neobavaisoflavone, corylifolinin, coryfolin and corylin noncompetitively inhibited the activity of CES1, and bavachinin competitively inhibited the activity of CES1. The inhibition kinetic parameters (K_i) were calculated to be 5.3, 9.4, 1.9, 0.7 and 0.5 μM for neobavaisoflavone, corylifolinin, coryfolin, corylin and bavachinin, respectively. In conclusion, the inhibition behavior of CES1 by the FP’s constituents was given in this article, indicating the possible adverse effects of FP through the disrupting CES1-catalyzed metabolism of endogenous substances and xenobiotics.     

Pharmacological pleiotropism of the human recombinant α1A-adrenoceptor: implications for α1-adrenoceptor classification

1. Three fully-defined α₁-adrenoceptors (α_1A, α_1B and α_1D) have been established in pharmacological and molecular studies. A fourth α₁-adrenoceptor, the putative α_1L-adrenoceptor, has been defined in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized α₁-adrenoceptors is not known.
2. In the present study, binding affinities were estimated by displacement of [³H]-prazosin in membrane homogenates of Chinese hamster ovary (CHO-K1) cells stably expressing the human α_1A-, α_1B- and α_1D-adrenoceptors and were compared with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)-stimulated accumulation of [³H]-inositol phosphates.
3. For the α_1A-adrenoceptor, binding studies revealed a pharmacological profile typical for the classically defined α_1A-adrenoceptor, such that prazosin, RS-17053, WB 4101, 5-methylurapidil, Rec 15/2739 and S-niguldipine all displayed subnanomolar affinity. A different profile of affinity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB 4101, 5-methylurapidil, RS-17053 and S-niguldipine showed 10 to 40 fold lower affinity than in membrane binding. However, affinity estimates were not ‘frameshifted'', as tamsulosin, indoramin and Rec 15/2739 yielded similar, high affinity estimates in binding and functional assays.
4. In contrast, results from human α_1B- and α_1D-adrenoceptors expressed in CHO-K1 cells gave antagonist affinity profiles in binding and functional assays that were essentially identical.
5. A concordance of affinity estimates from the functional (inositol phosphates accumulation) studies of the α_1A-adrenoceptor in CHO-K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative α_1L-adrenoceptor). These data show that upon functional pharmacological analysis, the cloned α_1A-adrenoceptor displays pharmacological recognition properties consistent with those of the putative α_1L-adrenoceptor. Why this profile differs from that obtained in membrane binding, and whether it explains the α_1L-adrenoceptor pharmacology observed in many native tissues, requires further investigation.

     

Triptolide suppresses IL-1βinduced chemokine and stromelysin-1 gene expression in human colonic subepithelial myofibroblasts

AIM: To examine the inhibitive effects of triptolide on the expression of IL-8, monocyte chemotactic protein (MCP)-1, and matrix metalloproteinases (MMP)-3 in subepithelial myofibroblasts (SEMF) stimulated with IL-1beta. METHODS: SEMF cultures were established from normal colons in patients who underwent gut resection for colorectal carcinoma. Chemokine and MMP-3 expressions were determined by ELISA and RT-PCR. The cytosolic amount of phosphorylation of I kappa B-alpha(p-I kappa B-alpha) was determined by Western blotting. The DNA binding capacity of NF-kappa B was evaluated by electrophoretic mobility shift assays. RESULTS: IL-1beta stimulated protein and mRNA expression of IL-8, MCP-1, and MMP-3 in SEMF. Triptolide inhibited these effects of IL-1beta in a dose-dependent manner. Mechanistic studies revealed that triptolide markedly decreased IL-1beta -induced NF-kappa B DNA binding capacity and cytosolic amount of p-I kappa B-alpha. These results showed that triptolide inhibited IL-1beta -induced chemokine and MMP-3 expression in SEMF through the NF-kappa B pathway. CONCLUSION: Triptolide inhibited IL-1beta -induced chemokine and MMP-3 expression in SEMF by preventing the phosphorylation of I kappa B-alpha.     

Quantification and distribution of α1-adrenoceptor subtype mRNAs in human proximal urethra

1. We performed RNase protection assays and in situ hybridization to investigate the ratio of the three α₁-adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross-sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra.
2. In situ hybridization studies showed no significant differences in the cross-sectional distribution of α₁-adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense.
3. Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α₁ drugs, α₁-selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.