雷公藤多苷对移植心冠状动脉硬化的抑制效果

目的　探讨雷公藤多苷(MT)对心移植后冠状动脉硬化及血小板衍化生长因子A (PDGF-A)表达的影响。方法　于大鼠腹腔内异位心脏移植术后分别应用MT ( 30mg·kg^-1·d^-1)(MT组=7例)或环胞素A (10mg·kg^-1·d^-1) (对照组=7例)。心移植后6 0d时观察比较组间移植心的冠状动脉硬化病变率及程度,排异反应及PDGF A的表达程度。结果　MT组的移植心冠状动脉硬化发生率和内膜增厚程度以及PDGF A的表达程度均显著低于对照组(P<0.01) ,而心肌排斥反应程度两组间无明显差异(P>0.05)。结论　MT可抑制移植心冠状动脉硬化病变,该效果可能与MT对移植心PDGF A表达的抑制作用有关     

褪黑素对多柔比星所致肾病的保护作用

本研究旨在观察褪黑素 ( MT)对大鼠“阿霉素肾病”的保护作用 .将所有受试动物分为 5组 ,即正常组 ,MT( d0 - d7,5mg·kg-1·d-1,ig)组 ,多柔比星模型组 ( d 1 ,Dox,5mg· kg-1,iv)组和MT( d 0 - d 7,0 .5,5mg· kg-1· d-1) + Dox( d 1 ,5mg· kg-1,iv)组 .检测大鼠 d7,d1 4,d2 1和 d2 8时尿蛋白 ,尿丙二醛排泄量和 d 2 8时血浆生化指标 .结果显示 ,Dox组大鼠呈典型的肾病综合征 ,MT+ Dox组大鼠尿蛋白减少 ,血浆蛋白明显回升 ,血脂降低 ;同时 ,Dox组动物尿丙二醛显著增加 ,MT+ Dox组丙二醛降低 .这些结果表明 ,MT可减轻“阿霉素肾病”大鼠肾损害 .     

褪黑激素对U_(14)的抑制作用

目的　探讨褪黑激素 (melatonin ,MT)对U14 的抑瘤作用及对常用化疗药环磷酰胺 (cytoxan ,CTX)的增效作用。方法　用小鼠移植性肿瘤模型 ,观察药物对荷U14 小鼠的肿瘤抑制率的影响。在可耐受剂量下治疗小鼠的实体瘤U14 ,观察药物对小鼠脾脏重量及肝脏组织结构的影响。结果　高剂量MT( 80mg·kg-1·d-1,sc)组对小鼠U14 有抑制作用 ,抑瘤率为 2 7 1% (P <0 0 5 )。高剂量组及低剂量MT( 4 0mg·kg-1·d-1,sc)组与CTX( 2 5mg·kg-1·d-1× 1,ip)合用对U14 的抑瘤作用可相加。高剂量MT与CTX合用对小鼠脾脏重量及肝脏组织结构均无明显影响。结论　MT有抑制U14 的作用 ,与CTX合用具有疗效相加的作用 ,毒性无明显增加。     

非清髓性造血干细胞移植临床研究

目的 探讨非骨髓清除性(非清髓)造血干细胞移植(NST)治疗血液病疗效及毒性。方法 16例血液病患者,供体为其HLA配型完全相合的同胞,供体动员方案为粒细胞集落刺激因子(G-CSF)300μg/12h×5天。预处理方案为氟拉达宾(Fludara)30mg·m-2·d-1×6天,马利兰(Bu)4mg·kg-1·d-1×2天;抗淋巴细胞免疫球蛋白(ALG)12～15mg·m-1·kg-1×4天,阿糖胞苷(Ara-C)500mg/d×4天或环磷酰胺(CTX)50mg·kg-1·d-1×2天)。单用环孢菌素A(CSA)3mg·kg-1·d-1预防移植物抗宿主病(GVHD)。移植后常规不用造血生长因子。结果 均顺利完成预处理及移植治疗。并发症发生率低,程度较轻,移植后+12天(中位时间)造血重建,14例已达供体完全或接近完全嵌合。随访120～485天,总存活率为87.5％。结论 NST能有效治疗多种血液病(包括恶性血液病),为血液病治疗提供又一安全有效的新策略。     

辛伐他汀对心肌梗死大鼠缺血心肌毛细血管新生的影响

目的 :评价辛伐他汀是否促进缺血心肌毛细血管新生。方法 :雄性Wistar大鼠 30只 ,成功复制心肌梗死模型 2 4h后随机分成辛伐他汀组、对照组和辛伐他汀 +N 硝酸 l 精氨酸甲酯 (l NAME)组 ,分别ip辛伐他汀 2mg·kg- 1·d- 1、生理盐水和辛伐他汀 2mg·kg- 1·d- 1+l -NAME15mg·kg- 1·d- 1。 4wk后应用免疫组化法及化学法检测各组大鼠缺血心肌中血管内皮细胞生长因子 (VEGF)蛋白质表达、一氧化氮 (NO )水平及毛细血管密度。结果 :与对照组相比辛伐他汀组大鼠缺血心肌中毛细血管密度显著增加 (196 3±s 10 8)·mm- 2 (P <0 .0 1) ,NO水平明显增高 (2 6± 5 )mmol·kg- 1(P <0 .0 1) ,但VEGF表达无明显改变 ;加用l NAME后 ,辛伐他汀的这些作用消失。结论 :辛伐他汀可促进大鼠缺血心肌毛细血管新生 ,NO可能介导了此作用。     

心得安防治儿童偏头痛32例疗效观察

目的观察心得安防治儿童偏头痛的疗效。方法选择儿童偏头痛患者64例随机分为两组,其中治疗组32例给予普萘洛尔(心得安)1~2mg·kg-1·d-1,分3次口服;对照组32例给予赛庚啶0·2~0·4mg·kg-1·d-1,分3次口服。疗程3个月,连续观察半年。观察头痛发作次数及持续时间的减少,程度的减轻及服药后反应。结果治疗组总有效率90·6%,对照组总有效率71·8%,两组比较差异有显著意义(P<0·01)。结论心得安防治儿童偏头痛安全有效。     

红霉素治疗以痰鸣音为主的小儿肺炎疗效观察

目的　比较红霉素与头孢类、青霉素类治疗以痰鸣音为主的小儿肺炎的疗效。方法　对 1 30例患儿随机分为治疗组 (A组 ) :5 0例 ,给予红霉素 2 5～ 30 mg·kg- 1· d- 1 ,静脉滴注 ;对照组 (B组 ) 4 0例给予头孢塞肟钠 5 0～1 0 0 mg· kg- 1 · d- 1 ) ,分两次静脉滴注 ;C组 4 0例给予益萨林 5 0～ 1 0 0 mg· kg- 1 · d- 1 ,分两次静脉滴注。结果　用药7d后 A组总有效率为 94 % ,B组总有效率为 72 .5 % ,C组总有效率为 6 2 .5 % ,A组与 B、C两组相比有显著差异性及非常显著差异性 (P<0 .0 5 ,P<0 .0 1 ) ,而且费用比较 ,A组与 B、C两组相比差异有非常显著性 (P<0 .0 1 )。结论　红霉素治疗以痰鸣音为主的小儿肺炎疗效优于头孢类及青霉素类 ,而且费用低 ,见效快 ,疗效高     

福辛普利、替米沙坦单用或联用对糖尿病模型大鼠颈动脉内膜球囊损伤后血管紧张素转换酶2的影响研究

目的:考察福辛普利、替米沙坦单用或联用对糖尿病模型大鼠颈动脉内膜球囊损伤后血管紧张素转换酶2(ACE2)的影响及其作用机制。方法:取大鼠36只,均分为正常对照组、糖尿病模型组(蒸馏水)、球囊损伤组(蒸馏水)、福辛普利组(10mg·kg-1·d-1)、替米沙坦组(0.8mg·kg-1·d-1)和联用组(福辛普利10mg·kg-1·d-1、替米沙坦0.8mg·kg-1·d-1),每组6只,除正常对照组外,其余各组大鼠腹腔注射链脲佐菌素(35mg·kg-1)建立2型糖尿病模型。建模成功后,除糖尿病模型组外,其余各组大鼠行颈动脉内膜球囊损伤手术,手术前、后分别灌服给予相应药物1、2周,正常对照组相同时间给予蒸馏水,检测各组大鼠颈动脉中ACE2mRNA及其蛋白表达。结果:与正常对照组比较,球囊损伤组大鼠ACE2 mRNA及其蛋白表达均明显降低(P<0.05);与球囊损伤组比较,福辛普利组大鼠ACE2 mRNA及其蛋白表达没有明显差异(P>0.05),替米沙坦组和联用组大鼠ACE2mRNA及其蛋白表达明显增加(P<0.05),但2组间无显著差异(P>0.05)。结论:福辛普利并不能促进替米沙坦增强ACE2的表达;替米沙坦可能是通过增加动脉内膜局部ACE2的表达来发挥血管保护作用。     

兰索拉唑对乙醇诱导大鼠胃黏膜损伤的保护作用及其机制

目的　研究兰索拉唑 (LP)对乙醇诱导大鼠胃黏膜损伤的保护作用 ,探讨胃泌素受体和环氧化酶 2 (COX 2 )表达在此过程中的作用。方法　大鼠ig给予LP 0 5、5、5 0mg·kg-1·d-1,或ig联合给予LP 5 0mg·kg-1·d-1和胃泌素受体拮抗剂AG 0 4 1R 3、10、30mg·kg-1·d-1,对照组ig给予羧甲基纤维素 (CMC) 2 5mg·kg-1·d-1,连续 14d。末次给药后 8h各组大鼠ig给予无水乙醇 1ml,观察胃损伤指数 (LI)及光镜下的胃黏膜病理学改变。酶免疫方法测定胃黏膜前列腺素E2 (PGE2 )水平 ,WesternBlot和免疫组化检测胃黏膜COX 2表达。评价特异性COX 2抑制剂NS 398对LP诱导的PGE2 合成及胃黏膜保护作用的影响。结果　在 0 5、5、5 0mg·kg-1LP组 ,LI分别为 (2 5 3± 0 33) %、(1 84±0 2 9) %和 (0 83± 0 12 ) % ,小于对照组 (3 6 5± 0 19) % (P<0 0 5 ) ;胃黏膜PGE2 含量分别为 (42 7± 32 ) ,(483± 12 1)和 (6 14± 82 ) pg·g-1wwt ,高于对照组 (2 6 6± 81) pg·g-1wwt(P <0 0 5 )。LP剂量依赖性地增加大鼠胃黏膜COX 2表达。然而 ,同时给予AG 0 4 1R阻断了LP诱导的胃黏膜保护作用、COX 2表达和PGE2 合成。NS 398抑制LP诱导的PGE2 合成及胃黏膜保护作用。结论　LP的胃黏膜保护作用与内源性胃泌素激活胃泌素受     

香椿叶总黄酮对糖尿病小鼠血糖的影响

目的研究香椿叶总黄酮对糖尿病小鼠血糖的影响。方法采用链脲佐菌素腹腔注射制备糖尿病小鼠模型,造模成功小鼠随机分为模型组、香椿叶总黄酮大剂量(800 mg·kg-1·d-1)、中剂量(400 mg·kg-1·d-1)和小剂量组(100 mg·kg-1·d-1),优降糖组(60mg·kg-1·d-1),连续给药30 d后,观察血糖的变化。结果香椿总黄酮可明显降低糖尿病小鼠的血糖。结论香椿叶总黄酮有明显的降糖作用,为临床上防治糖尿病提供了理论依据。     

Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors

In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.     

Influence of iron (56Fe2O3 or 54Fe2O3) in the upregulation of cytochrome P4501A1 by benzo[a]pyrene in the respiratory tract of Sprague-Dawley rats

Any influence of iron in polycyclic aromatic hydrocarbon (PAH)/iron oxide mixtures on the capacity of PAHs to induce metabolizing enzymes will be one of the ways that iron oxides can affect PAH carcinogenicity. Because cytochromes P450 (CYPs) are haemoproteins, it will be of interest to investigate the possible involvement of Fe(2)O(3) in benzo[a]pyrene (BaP)/Fe(2)O(3) mixtures on the induction of CYP1A1 enzymes in the lung. Male Sprague-Dawley rats were instilled intratracheally with haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3), 3 mg), BaP (3 mg) or BaP (3 mg) coated onto haematite ((56)Fe(2)O(3) or (54)Fe(2)O(3)) particles (3 mg). Firstly, mRNA expressions of cyp1a1 were studied. Secondly, protein concentrations and catalytic activities (7-ethoxyresorufin O-deethylase: EROD) of CYP1A1 were determined. Thirdly, (54)Fe from BaP/(54)Fe(2)O(3) mixtures in microsomal proteins was studied using time-of- flight laser microprobe mass spectrometry (ToF-LMMS). Statistically significant increases in mRNA expressions, protein concentrations and catalytic activities of CYP1A1 were observed in animals exposed to BaP, to BaP coated onto (56)Fe(2)O(3) particles or to BaP coated onto (54)Fe(2)O(3) particles versus controls. Both of the BaP/Fe(2)O(3) mixtures induced higher CYP1A1 protein levels and EROD activities than BaP alone. Iron oxide particles per se did not affect mRNA levels of cyp1a1 but only enhanced BaP-mediated increases of CYP1A1 protein levels and activity. The ToF-LMMS spectrum pro fi les showed that the (54)Fe/(56)Fe ratio in the microsomes of BaP coated onto (54)Fe(2)O(3) particle-instilled animals was 1.3 instead of the theoretical ratio (i.e. 0.063) observed in BaP coated onto (56)Fe(2)O(3) particle-instilled animals. Taken together, these novel data support the hypothesis that the Fe(2)O(3)-induced increases of the metabolic activation of BaP might rely on the property of Fe(2)O(3) particles to enhance the BaP-induced translation rate of the cyp1a1 gene into functional haemoproteins.     

S-allylcysteine scavenges singlet oxygen and hypochlorous acid and protects LLC-PK(1) cells of potassium dichromate-induced toxicity.

It has been found that S-allylcysteine (SAC), a garlic-derived compound, has in vivo and in vitro antioxidant properties. In addition, it is known that SAC is able to scavenge different reactive oxygen or nitrogen species including superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH()), and peroxynitrite anion (ONOO(-)) although the IC(5O) values for each reactive species has not been calculated and the potential ability of SAC to scavenge singlet oxygen ((1)O(2)) and hypochlorous acid (HOCl) has not been explored. The purposes of this work was (a) to explore the potential ability of SAC to scavenge (1)O(2) and HOCl, (b) to further characterize the O(2)(-), H(2)O(2), OH(), and ONOO(-) scavenging ability of SAC by measuring the IC(50) values using in vitro assays, and (c) to explore the potential ability of SAC to ameliorate the potassium dichromate (K(2)Cr(2)O(7))-induced cytotoxicity in LLC-PK1 cells in which oxidative stress is involved. The scavenging activity was compared against the following reference compounds: N-acetylcysteine for O(2)(-), sodium pyruvate for H(2)O(2), dimethylthiourea for OH(), lipoic acid and glutathione for (1)O(2), lipoic acid for HOCl, and penicillamine for ONOO(-). It was found that SAC was able to scavenge concentration-dependently all the species assayed with the following IC(5O) (mean+/-SEM, mM): O(2)(-) (14.49+/-1.67), H(2)O(2) (68+/-1.92), OH() (0.68+/-0.06), (1)O(2) (1.93+/-0.27), HOCl (2.86+/-0.15), and ONOO(-) (0.80+/-0.05). When the ability of SAC to scavenge these species was compared to those of the reference compounds it was found that the efficacy of SAC (a) to scavenge O(2)(-), H(2)O(2), OH(), and ONOO(-) was lower, (b) to scavenge HOCl was similar, and (c) to scavenge (1)O(2) was higher. In addition, it was found that SAC was able to prevent K(2)Cr(2)O(7)-induced toxicity in LLC-PK1 cells in culture. It was showed for the first time that SAC is able to scavenge (1)O(2) and HOCl and to ameliorate the K(2)Cr(2)O(7)-induced toxicity.     

Adsorption of metal ions under chemically modified silica gel with the presence of Aza Crown Ether at physiological pH

The competitive adsorption of Zn(II), Cu(II), Co(II) and Ni(II) in Zn(II)–Cu(II), Zn(II)–Co(II), Zn(II)–Ni(II), Cu(II)–Co(II), Cu(II)–Ni(II) and Co(II)–Ni(II) binary systems from aqueous solutions by Kryptofix 22 (Aza Crown Ether) functionalized silica gel (SIL-Crown) adsorbent were investigated. SIL-Crown adsorbent was characterized by FT-IR. The results exhibited that SIL-Crown adsorped Zn (II) in the systems of Zn(II)–Co(II) and Zn(II)–Ni(II). Thus SIL-Crown is favorable and useful for the removal of Zn(II), and high adsorption makes it a promising candidate for Zn(II) uptake.     

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.     

Direct enantioselective determination of (R)- and (S)-propranolol in human plasma. Application to pharmacokinetic studies

In order to examine possible drug interactions of (R)- and (S)-propranolol a randomized, double blind crossover study has been performed, administering orally single doses of 40 mg (R,S)- and of 20 mg (S)-propranolol.HCl three times daily over a week to reach steady state conditions. After the first single dose of 40 mg (R,S)-propranolol.HCl, the AUC_o−t8 and C_max values of the (S)-isomer were greater than those of the (R)-isomer: the ratio of AUC_(s) over AUC_(R) was 1.77 (P < 0.05) and that of C_max 1.57 (P < 0.01). When (S)-propranolol.HCl was given as a single 20 mg dose, the AUC_(s) value was a factor of 0.55 lower than after administration of 40 mg (R,S)-propranolol.HCl. At steady state, the AUC of (S)-propranolol was 1.52 times higher (P < 0.01) than that of the (R)-isomer after administration of 40 mg racemate, and comparing the (S)-isomer, the ratio was 1.21. Following administration of the first single dose of 40 mg of the racemate, the mean (SD) clearance of the (R)- and (S)-isomers was 110 (84) and 61 (37) ml min⁻¹ kg⁻¹, respectively; at steady state these values were 89 (55) and 57 (37) ml min⁻¹ kg⁻¹, respectively. Respective values for (S)-propranolol after single isomer administration (20 mg) were 86 (36) and 57 (25) ml min⁻¹ kg⁻¹ in single dose and steady state situations. The data are based on the quantitative analysis of (R)- and (S)-propranolol in plasma. A sensitive enantioselective LC-bioassay based on the formation of the (R)- and (S)-propranolol-oxazolidine-2-one and resolution of these derivatives on a (R,R)-dinitrobenzoyl-diaminocyclohexane ((R,R)-DNB-DACH) chiral stationary phase was developed, using dichloromethane—methanol (99.75:0.25, v/v) as mobile phase, with fluorimetric detection.     

Direct cardiac effects of As2O3 in rabbits: evidence of reversible chronic toxicity and tissue accumulation of arsenicals after parenteral administration

Although parenteral administration of As(2)O(3) is highly effective in the treatment of acute promyelocytic leukemia, cardiac toxicity has been reported. This study employed Langendorff perfusion to determine the direct effects of As(2)O(3) in the electrophysiological properties of rabbit hearts after acute or chronic As(2)O(3) treatment (0.2 mg/kg/day iv for 30 days). Tissue accumulations of arsenicals and pathological changes as well as the reversibility of chronic As(2)O(3) effects were assessed. We found that cardiac conduction and repolarization were not altered whatsoever after acute As(2)O(3) treatment at clinically relevant (1, 3, and 10 microM) and higher (30 microM) doses. Nevertheless, an extremely high concentration of As(2)O(3) (300 microM) prolonged the corrected QT interval. Subsequent to chronic As(2)O(3) administration and with 30 microM As(2)O(3) via Langendorff perfusion, polymorphic ventricular tachycardia was observed (1/7, 14%). Corrected QT interval was prolonged, while basic cycle length was shortened. Significant accumulation of arsenicals in the cardiac tissue was found, but without any pathological changes. After As(2)O(3) was discontinued for 30 days, the chronic As(2)O(3) -induced electrophysiological changes improved, no ventricular arrhythmia was noted, and the tissue concentration of arsenicals decreased considerably. We therefore conclude that, although no immediate cardiac effects were discemable at clinically relevant doses, an extremely high concentration of As(2)O(3) could prolong ventricular repolarization. Chronic As(2)O(3) treatment resulted in a prolonged ventricular repolarization, in association with arsenicals accumulation and with risk of ventricular tachycardia. These chronic cardiac toxicities and the tissue accumulation of arsenicals were, however, partially reversible after cessation of As(2)O(3).     

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.     

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets

Redox regulation is important for the modulation of cytosolic Ca(2+) concentration. Hence, we have investigated the effect of H(2)O(2) on store-mediated Ca(2+) entry (SMCE). In fura-2-loaded human platelets treatment with H(2)O(2) resulted in a concentration-dependent increase in Ca(2+) release from intracellular stores, while the effect on Ca(2+) entry was biphasic. In addition, 1mM H(2)O(2) reduced SMCE induced by agonists. The inhibitory effect of 1mM H(2)O(2) was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10microM H(2)O(2) and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1mM H(2)O(2) shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H(2)O(2)-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1mM H(2)O(2) significantly increased store depletion-induced p60(src) activation. We conclude that H(2)O(2) exerted a biphasic effect on SMCE. The inhibitory role of high H(2)O(2) concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.     

Pharmacological characterization of human NPFF(1) and NPFF(2) receptors expressed in CHO cells by using NPY Y(1) receptor antagonists

Neuropeptide FF (NPFF) belongs to an opioid-modulatory system including two precursors (pro-NPFF(A) and pro-NPFF(B)) and two G-protein coupled receptors (NPFF(1) and NPFF(2)). The pharmacological and functional profiles of human NPFF(1) and NPFF(2) receptors expressed in Chinese hamster ovary (CHO) cells were compared by determining the affinity of several peptides derived from both NPFF precursors and by measuring their abilities to inhibit forskolin-induced cAMP accumulation. Each NPFF receptor recognizes peptides from both precursors with nanomolar affinities, however, with a slight preference of pro-NPFF(A) peptides for NPFF(2) receptors and of pro-NPFF(B) peptides for NPFF(1) receptors. BIBP3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide) and BIBO3304 ((R)-N(2)-(diphenylacetyl)-N-[4-(aminocarbonylaminomethyl)-benzyl]-argininamide trifluoroacetate), two selective neuropeptide Y (NPY) Y(1) receptor antagonists, display relative high affinities for NPFF receptors and exhibit antagonist properties towards hNPFF(1) receptors. The structural determinants responsible for binding of these molecules to NPFF receptors were investigated and led to the synthesis of hNPFF(1) receptor antagonists with affinities from 40 to 80 nM. Our results demonstrate differences in pharmacological characteristics between NPFF(1) and NPFF(2) receptors and the feasibility of subtype-selective antagonists.