Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)₂D₃- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)₂D₃ or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)₂D₃- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.     

慢性阻塞性肺疾病患者血清1,25-二羟维生素D3与病情程度的关系研究

目的对慢性阻塞性肺疾病(COPD)患者血清1,25-二羟维生素D₃[1,25(OH)₂D₃]进行检测,同时分析其运动耐力与肺功能的关系,以探讨COPD患者检测血清1,25(OH)₂D₃的临床意义。方法选取90例COPD患者作为COPD组,根据维生素D标准将COPD组分为1,25(OH)₂D₃正常组(9例)和1,25(OH)₂D₃缺乏组(81例)。随机选取同期进行体检的60例健康者作为对照组。检测所有研究对象的血清1,25(OH)₂D₃水平及肺功能指标。比较COPD患者1,25(OH)₂D₃正常组与1,25(OH)₂D₃缺乏组、1,25(OH)₂D₃缺乏组与对照组的1,25(OH)₂D₃水平、第1秒用力呼气容积(FEV1)、FEV1占预计值百分比(FEV1%)、FEV1占用力肺活量的百分比(FEV1/FVC),以及不同COPD全球策略修订版(GLOD)分级患者1,25(OH)₂D₃的水平。结果1,25(OH)₂D₃正常组患者的1,25(OH)₂D₃水平(29.4±10.5)ng/ml、FEV1/FVC(75.33±5.67)%均高于1,25(OH)₂D₃缺乏组的(17.4±4.3)ng/ml、(66.44±9.81)%,差异均具有统计学意义(P<0.05)。两组FEV1、FEV1%水平比较,差异均无统计学意义(P>0.05)。1,25(OH)₂D₃缺乏组1,25(OH)₂D₃水平(17.4±4.3)ng/ml、FEV1(2.13±0.46)L、FEV1%(64.33±6.58)%、FEV1/FVC(66.44±9.81)%低于对照组的(31.2±2.3)ng/ml、(2.94±0.58)L、(81.69±12.47)%、(96.18±3.69)%,差异均具有统计学意义(P<0.05)。Ⅰ、Ⅱ、Ⅲ、Ⅳ级GOLD分级患者的1,25(OH)₂D₃水平分别为(29.4±10.5)、(22.8±7.6)、(13.5±3.5)、(12.1±4.3)ng/ml,Ⅰ级患者1,25(OH)₂D₃水平高于Ⅱ、Ⅲ、Ⅳ级患者,Ⅱ级患者高于Ⅲ、Ⅳ级患者,差异均具有统计学意义(P<0.05)。Ⅲ级与Ⅳ级患者1,25(OH)₂D₃水平比较,差异无统计学意义(P>0.05)。结论COPD患者的血清1,25(OH)₂D₃水平显著低于健康人群,且1,25(OH)₂D₃缺乏的患病率更高;1,25(OH)₂D₃缺乏COPD患者的肺功能较1,25(OH)₂D₃正常COPD患者更低,并且随着COPD严重程度的增加,1,25(OH)₂D₃缺乏的程度加重。     

Differential involvement of protein kinase C in human promyelocytic leukemia cell differentiation enhanced by artemisinin

Artemisinin, a sesquiterpene lactone endoperoxide that exists in several medicinal plants, is a well-known anti-malarial agent. In this report, we investigated the effect of artemisinin on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Artemisinin markedly increased the degree of HL-60 leukemia cell differentiation when simultaneously combined with low doses of 1 alpha,25-dihydoxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (all-trans RA). Artemisinin by itself had very weak effects on the differentiation of HL-60 cells. Cytofluorometric analysis and cell morphologic studies indicated that artemisinin potentiated 1,25-(OH)(2)D(3)-induced cell differentiation predominantly into monocytes and all-trans RA-induced cell differentiation into granulocytes, respectively. Extracellular-regulated kinase (ERK) inhibitors markedly inhibited HL-60 cell differentiation induced by artemisinin in combination with 1,25-(OH)(2)D(3) or all-trans RA, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitors did not. Particularly, protein kinase C (PKC) inhibitors inhibited HL-60 cell differentiation induced by artemisinin in combination with 1,25-(OH)(2)D(3) but not with all-trans RA. Artemisinin enhanced PKC activity and protein level of PKC beta I isoform in only 1,25-(OH)(2)D(3)-treated HL-60 cells. Taken together, these results indicate that artemisinin strongly enhanced 1,25-(OH)(2)D(3)- and all-trans RA-induced cell differentiation in which PKC is differentially involved in arteminisin-mediated enhancement of leukemia cell differentiation.     

1,25-二羟基维生素D3抑制脂肪细胞分化作用的研究

【摘要】目的研究1,25-二羟基维生素D3[1,25(OH)₂D₃]对脂肪细胞分化的影响及其机制。方法以间充质干细胞系C3H10T1/2为研究对象,将其分为6组,分别为对照组、诱导分化组和4个不同剂量的1,25(OH)₂D₃处理组。其中对照组加入溶媒,诱导分化组加入脂肪细胞诱导分化试剂,1,25(OH)₂D₃处理组除了加入脂肪细胞诱导分化试剂外,予以10^-9、10^-8、10^-7、10^-6mol/L 1,25(OH)₂D₃处理。处理后5d进行油红O染色,RT-PCR检测脂肪细胞特异性转录因子和Wnt/β-catenin信号通路相关因子表达水平,Western blot检测β-catenin蛋白水平。结果1,25(OH)₂D₃能够强烈抑制脂肪细胞生成,阻断脂肪细胞特异性转录因子过氧化物酶体增殖物激活受体（PPAR）γ、CCAAT增强子结合蛋白（C/EBP）α及脂肪细胞表征因子aP2mRNA表达,下调Wnt/β-catenin信号通路抑制因子分泌性卷曲相关蛋白1（sFRP1）mRNA,上调β连环素（β-catenin）蛋白水平。结论1,25(OH)₂D₃强烈抑制脂肪细胞分化,该作用可能与其激活Wnt/β-catenin信号通路有关。     

Synergistic induction of 1,25-dihydroxyvitamin D(3)- and all-trans-retinoic acid-induced differentiation of HL-60 leukemia cells by yomogin,a sesquiterpene lactone from Artemisia princeps

Many anti-inflammatory agents are known to significantly enhance the terminal differentiation of some cancer cells such as leukemia cells. In this study, the effect of yomogin, a eudesmane sesquiterpene lactone isolated from Artemisia princeps with anti-inflammatory activity, was investigated in human promyelocytic leukemia HL-60 cells. Yomogin by itself induced small increases in cell differentiation, with less than 19 % of the cells attaining a differentiated phenotype. Importantly, yomogin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either 5 nM 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2) D(3)] or 50 nM all- trans retinoic acid (all- trans RA). Cytofluorometric analysis and morphologic studies indicated that the combinations of yomogin and 1,25-(OH)(2) D(3) stimulated differentiation to monocytes whereas the combinations of yomogin and all- trans RA stimulated differentiation to granulocytes. These results suggest that yomogin may be useful in combination with 1,25-(OH)(2) D(3) or all- trans-RA in the differentiation therapy for myeloid leukemias. Abbreviations. 1,25-(OH)(2) D(3) :1,25-dihydroxyvitamin D(3) FITC:fluorescein isothiocyanate NBT:nitroblue tetrazolium RA:retinoic acid PE:phytoerythrin     

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by silibinin: involvement of protein kinase C

The effect of silibinin, an active component of Silybum marianum, on cellular differentiation was investigated in the human promyelocytic leukemia HL-60 cell culture system. Treatment of HL-60 cells with silibinin inhibited cellular proliferation and induced cellular differentiation in a dose-dependent manner. Cytofluorometric analysis and morphologic studies indicated that silibinin induced differentiation of HL-60 cells predominantly into monocytes. Importantly, strongly synergistic induction of differentiation into monocytes was observed when silibinin was combined with 5 nM 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], a well-known differentiation inducer of HL-60 cells into the monocytic lineage. Silibinin enhanced protein kinase C (PKC) activity and increased protein levels of both PKCalpha and PKCbeta in 1,25-(OH)(2)D(3)-treated HL-60 cells. PKC and extracellular signal-regulated kinase (ERK) inhibitors significantly inhibited HL-60 cell differentiation induced by silibinin alone or in combination with 1,25-(OH)(2)D(3), indicating that PKC and ERK may be involved in silibinin-induced HL-60 cell differentiation.     

Synthesis of diacetoxy acetal derivatives of santonin and their enhancing effects on HL-60 leukemia cell differentiation

Several diacetoxy acetal analogues have been synthesized from santonin and assessed for their ability of inducing or enhancing the differentiation of human HL-60 leukemia cells. The compounds themselves had little effect on HL-60 cell differentiation. However, three analogues, 2a, 3a, and 5b, synergistically enhanced 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced HL-60 cell differentiation when combined with 5 nM of dihydroxyvitamin D3 [1,25-(OH)2D3], a well-known differentiation inducer. Especially, the compound 5b profoundly enhanced the 1,25-(OH)2D3]-induced HL-60 cell differentiation.     

Potentiation of 1,25-dihydroxyvitamin D(3)-induced differentiation of human promyelocytic leukemia cells into monocytes by costunolide,a germacranolide sesquiterpene lactone

Costunolide, a germacranolide sesquiterpene lactone that exists in several medicinal plants, is known to be a possible anti-cancer and chemopreventive agent for tumorigenesis. In this report, we investigated the effect of costunolide on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Costunolide markedly increased the degree of HL-60 leukemia cell differentiation when simultaneously combined with 5nM 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). Costunolide by itself had very weak effects on the differentiation of HL-60 cells. Cytofluorometric analysis and cell morphologic studies indicated that costunolide potentiated 1,25-(OH)(2)D(3)-induced cell differentiation predominantly into monocytes. Inhibitors for PKC, PI3-K, and ERK markedly inhibited HL-60 cell differentiation induced by costunolide in combination with 1,25-(OH)(2)D(3). In addition, pretreatment of HL-60 cells with costunolide before the 1,25-(OH)(2)D(3) addition also potentiated cell differentiation in a concentration- and time-dependent manner, and the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-kappaB-binding activity by costunolide. These results indicate that PKC, PI3-K, ERK and NF-kappaB may be involved in 1,25-(OH)(2)D(3)-mediated cell differentiation enhanced by costunolide.     

Evaluation of the role of nicotinic acetylcholine receptor subtypes and cannabinoid system in the discriminative stimulus effects of nicotine in rats

Male Wistar rats were trained to discriminate (−)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025–0.4 mg/kg; combination studies): the ₄β₂ nicotinic acetylcholine receptor subtype antagonist dihydro-β-erythroidine (DHβE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the ₇ nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the ₄β₂ nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB₁ receptor antagonist/partial agonist rimonabant, the cannabinoid CB₂ receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB_1/2 receptor agonists (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive ₇ nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404 + anandamide or URB 597 + anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHβE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404 + anandamide or URB 597 + anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of ₄β₂ (but not of ₇) subunits. Furthermore, we excluded the involvement of either cannabinoid CB₁ and CB₂ receptors or increases in the endocannabinoid tone in the nicotine discrimination.     

Intrinsic sympathomimetic activity of beta-adrenoceptor antagonists: down-regulation of cardiac beta 1- and beta 2-adrenoceptors

Prolonged treatment of cultured rat heart muscle cells containing β₁- and non-muscle cells containing β₂-adrenoceptors with β-adrenoceptor antagonists devoid of intrinsic sympathomimetic activity had no effect on β-adrenoceptor density. In contrast, antagonists with intrinsic sympathomimetic activity decreased β-adrenoceptor density and response (adenylate cyclase stimulation) in both heart muscle (β₁) and non-muscle cells (β₂) by a maximum of about 50%. An even larger down-regulation of β-adrenoceptors and loss of receptor-stimulated adenylate cyclase activity was induced by the full endogenous agonist, noradrenaline, with the β-adrenoceptors of heart muscle cells (β₁) being much more sensitive to the β₁-selective noradrenaline than the heart non-muscle cell β₂-adrenoceptors. When combined with noradrenaline, the antagonists with intrinsic sympathomimetic activity prevented the action of noradrenaline at both β₁- and β₂-adrenoceptors, thereby leading to an apparent up-regulation of receptor density and response. This apparent reversal from an agonist to an antagonist action was observed at much lower concentrations of noradrenaline at β₁- than at β₂-adrenoceptors. The data presented indicate that the β-adrenoceptor antagonists with intrinsic sympathomimetic activity, but not those without, upon prolonged treatment decrease the density and responsiveness of both β₁- and β₂-adrenoceptors in cultured rat heart cells. This suggests that the intrinsic sympathomimetic activity of these agents is not a subtype-selective component. Furthermore, the agonist and antagonist activity of these agents apparently depends on the concomitant presence of an endogenous full agonist and an its own affinity and that of the partial agonist for the β-adrenoceptor subtype.     

Synthesis of <Emphasis Type="Italic">C</Emphasis><Subscript>6</Subscript>-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)₂D₃ or ATRA and chemotherapy, adverse effects 1,25-(OH)₂D₃ or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C₆-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C₆-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6β-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)₂D₃ or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)₂D₃ or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.     

Roles of metabotropic glutamate receptor subtypes in modulation of pentylenetetrazole-induced seizure activity in mice

The anticonvulsant or proconvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in a chemoconvulsant model using pentylenetetrazole (PTZ). Mice received mGluR ligands by intracerebroventricular (i.c.v.) infusion prior to a subcutaneous injection of PTZ and the latency to onset of tonic convulsions was recorded. The group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA) dose-dependently antagonised PTZ-induced seizures with a mean ED₅₀ value of 465 nmol. In contrast, the selective group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine [(S)-DHPG], was proconvulsive and decreased the PTZ-induced seizure latency (ED₅₀=60 nmol i.c.v.). A selective agonist of group II mGluRs, (1S,3S)-1-aminocyclopentane dicarboxylic acid [(1S,3S)-ACPD], was proconvulsive but did not affect PTZ-induced seizure latency. Moreover, the proconvulsant effect of (1S,3S)-ACPD was not blocked by the mGluR2 antagonist, -methylserine-O-phosphate monophenyl ester but was blocked by AIDA suggesting the involvement of group I mGluRs. (2S,1′S,2′S,3′R)-2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG-IV), which is a potent mGluR2 antagonist and a group III mGluR agonist at higher doses, increased the PTZ-induced seizure latency (ED₅₀=51 nmol) and this effect was fully reversed by the group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4). Similarly, the group III mGluR agonist 1-amino-3-(phosphonomethylene)cyclobutanecarboxylate (cyclobutylene-AP5) increased the PTZ-induced seizure latency (ED₅₀=12 nmol) in a MAP4-sensitive manner. Collectively, these data suggest that mGluR ligands modulate PTZ-induced seizure activity in mice by either antagonizing group I mGluRs or activating group III mGluRs.     

Characterization of two novel σ receptor ligands: antidystonic effects in rats suggest σ receptor antagonism

The novel σ receptor ligands, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine (BD1063), were characterized in rats using binding assays and behavioral studies. In radioligand binding studies, the novel ligands showed marked selectivity for σ binding sites, generally having a 100-fold or better affinity for σ sites compared to nine other tested receptors (opiate, phencyclidine, muscarinic, dopamine, ₁-, ₂-, β-adrenoceptor, 5-HT₁, 5-HT₂); the only exception was the affinity of BD1047 for β-adrenoceptors. Competition assays further revealed that the drugs interacted with both σ₁ and σ₂ binding sites. Although both drugs had preferential affinities for σ₁ sites, BD1047 exhibited a higher affinity for σ₂ sites than BD1063. In behavioral studies, BD1047 and BD1063 had no effects on their own when unilaterally microinjected into the red nucleus of rats, but both compounds attenuated the dystonia produced by the high affinity σ ligands, di-o-tolylguanidine (DTG) and haloperidol. BD1047 and BD1063 dose-dependently attenuated the dystonia produced by DTG, suggesting a receptor-mediated mechanism, and the dose curve for DTG was shifted to the right in the presence of the novel ligands. BD1047 and BD1063 appear to act as antagonists at σ sites and may represent promising new tools for probing other functional effects associated with σ binding sites.     

The effects of cyclopentane and cyclopentene analogues of GABA at recombinant GABA(C) receptors.

The pharmacological effects of the enantiomers of cis-3-aminocyclopentanecarboxylic acids ((+)- and (−)-CACP), the enantiomers of trans-3-aminocyclopentanecarboxylic acids ((+)- and (−)-TACP), and the enantiomers of 4-aminocyclopent-1-ene-1-carboxylic acids ((+)- and (−)-4-ACPCA) were studied on human homomeric ρ₁ and ρ₂ GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp methods. These compounds are conformationally restricted analogues of γ-aminobutyric acid (GABA) held in a five-membered ring. (+)-TACP (EC₅₀ (ρ₁)=2.7±0.2 μM; EC₅₀ (ρ₂)=1.45±0.22 μM), (+)-CACP (EC₅₀ (ρ₁)=26.1±1.1 μM; EC₅₀ (ρ₂)=20.1±2.1 μM) and (−)-CACP (EC₅₀ (ρ₁)=78.5±3.5 μM; EC₅₀ (ρ₂)=63.8±23.3 μM) were moderately potent partial agonists at ρ₁ and ρ₂ GABA_C receptors, while (−)-TACP (100 μM inhibited 56% and 62% of the current produced by 1 μM GABA at ρ₁ and ρ₂ receptors, respectively) was a weak partial agonist with low intrinsic activity at these receptors. In contrast, (+)-4-ACPCA (K_i (ρ₁)=6.0±0.1 μM; K_i (ρ₂)=4.7±0.3 μM) did not activate GABA_C ρ₁ and ρ₂ receptors but potently inhibited the action of GABA at these receptors, while (−)-4-ACPCA had little effect as either an agonist or an antagonist. The affinity order at both GABA_C ρ₁ and ρ₂ receptors was (+)-TACP>(+)-4-ACPCA(+)-CACP>(−)-CACP(−)-TACP(−)-4-ACPCA. This study shows that the cyclopentane and cyclopentene analogues of GABA affect GABA_C receptors in a unique manner, defining a preferred stereochemical orientation of the amine and carboxylic acid groups when binding to GABA_C receptors. This is exemplified by the partial agonist, (+)-TACP, and the antagonist, (+)-4-ACPCA.     

Patensin-induced apoptosis is accompanied by decreased bcl-2 expression and telomerase activity in HL-60 cells

The present study aimed to investigate the effect of telomerase activity in patensin-induced apoptosis and the regulation of B cell leukemia/lymphoma 2 (bcl-2) gene expression in human leukemia HL-60 cells. Apoptosis of HL-60 cells was induced by patensin (100 μmol L_-1) for 3, 6, 12 and 24 h. Apoptosis and bcl-2 were determined by flow cytometry analysis. A polymerase-chain-reaction-based telomeric repeat amplification protocol assay was used to detect the telomerase activity. Patensin induced growth arrest and apoptotic cell death in HL-60 cells. The telomerase activity was inhibited in a time-dependent manner during the patensin-induced apoptosis of HL-60 cells, and the expression of bcl-2 was progressively down-regulated by patensin. Inhibition of the telomerase activity of HL-60 cells was closely related to the patensin-induced apoptosis. The present results indicate that inhibition in telomerase and reduced bcl-2 gene expression may play a role in the patensin-induced apoptosis of HL-60 cells.     

Direct Activation of GABAA Receptors by Loreclezole, an Anticonvulsant Drug with Selectivity for the β-Subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates γ-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor β-subunits. A similar selectivity for GABA_A receptor β-subunits is apparent for the direct activation of receptor-operated Cl⁻ channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABA_A receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding by up to 28% (at 5 μM) and 80% (at 10 μM), respectively. Higher concentrations (50–100 μM) of both compounds inhibited [³⁵S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [³⁵S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50–100 μM, loreclezole induced inward Cl⁻ currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABA_A receptors, comprised of ₁-, β₂- and γ_2S-subunits. At 100 μM, the current evoked by loreclezole was 26% of that induced by 5 μM GABA. The current evoked by 100 μM propofol was 98% of that induced by 5 μM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABA_A receptors containing the β₂-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABA_A receptors containing the β₁-subunit may be responsible for its lack of hypnotic effect. © 1997 Published by Elsevier Science Ltd. All rights reserved.     

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABA_A receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABA_A (₁β₂γ_2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABA_A receptors. The GABA enhancing action of E-6375 on recombinant GABA_A receptors was unaffected by the subtype of the isoform (i.e. _xβ₂γ_2L; x=1–3) within the receptor, but was increased by the omission of the γ_2L subunit. Receptors incorporating β₂, or β₃, subunits were more sensitive to modulation by E-6375 than those containing the β₁ subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the β-subunit. The various in vivo actions of general anaesthetics may be mediated by GABA_A receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABA_A receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.     

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells

When CCRF-CEM cells were incubated with 5–40 μM CdCl_2, apoptosis was observed most clearly at 10 μM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl₂ exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 μM CdCl_2, but higher than 20 μM CdCl₂ was required for the clear phosphorylation of JNK. In the time–course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl₂ exposure. The in vitro activities of MAPKs also increased in response to CdCl₂ exposure. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl₂-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca²⁺ but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), suppressed CdCl₂-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl_2, and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl₂ exposure in this human T cell line.     

Puerarin protects rat pancreatic islets from damage by hydrogen peroxide

Periodate-oxidized 2′,3′-dialdehyde ATP (oxidized ATP) has been used extensively as a selective antagonist at P2X₇ receptors, although P2X₇-independent actions on pro-inflammatory cytokine release have also been reported. Because P2X₇ receptors in astrocytes have been suggested as potential targets of anti-inflammatory drug therapy, we examined the effect of oxidized ATP on β-actin expression and superoxide production of RBA-2 type-2 astrocytes known to possess P2X₇ receptors. Oxidized ATP per se decreased β-actin expression time and dose dependently. Treatment with oxidized ATP for 8 h caused an approximately 50% decrease in β-actin expression whereas other P2 receptor antagonists, brilliant blue G (BBG), suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), were not effective. In addition, oxidized ATP per se decreased the intracellular superoxide concentration, whereas ATP and the P2X₇ receptor-selective agonist 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) stimulated intracellular superoxide production, an effect inhibited by oxidized ATP. In addition, oxidized ATP neither affected cellular viability nor affected interleukin-1β, converting enzyme (ICE)-like protease activity in these astrocytes. To further elucidate the mechanism, the effects of oxidized ATP on intracellular superoxide concentration and β-actin expression were examined in a P2X₇ receptor-negative astrocyte cell line, IA-1g1. Oxidized ATP-induced a time-dependent decrease in intracellular superoxide concentration whereas oxidized ATP had no effect on β-actin expression. Nevertheless, oxidized ATP altered f-actin cytoskeleton arrangement in IA-1g1 astrocytes. Taken together, these results indicate that oxidized ATP per se caused a cell specific decrease in β-actin expression in RBA-2 type-2 astrocytes. In addition, oxidized ATP decreased intracellular superoxide concentrations and altered f-actin cytoskeleton arrangement in both P2X₇ receptor-positive and -negative astrocytes. Thus, we conclude from these results that the effects of oxidized ATP on actin and superoxide are mediated through mechanisms that are at least in part, independent of P2X₇ receptors.