卒中易感型自发性高血压大鼠血管平滑肌收缩装置对Ca^2＋敏感性变…

比较卒中易感型自发性高血压大鼠与常压大鼠基底动脉和尾动脉平滑肌收缩装置对Ｃａ＾２＋敏感性的差异。采用血管平滑肌细胞膜化学高通透技术并结合细胞钙钳制技术，使上述血管肌条平滑肌细胞膜具有高通透性，并使胞内Ｃａ＾２＋固定于一定水平，然后制作神经鞘氨醇预温浴后的肌条ｐＣａ－张力曲线，比较ＳＨＲｓｐ和Ｗｉｓａｔｒ大鼠ＢＡ和ＣＡ平滑肌在不同处理时的ｐＣａ５０值。     

交感神经损毁对自发性高血压大鼠血压和红细胞膜ATP酶活性…

自发性高血压大鼠（ＳＨＲ）红细胞膜Ｎａ＾＋，Ｋ＾＋－ＡＴＰ酶和Ｃａ＾２＋，Ｍｇ＾２＋－ＡＴＰ酶活性分别高于和低于Ｗｉｓｔａｒ Ｋｙｏｔｏ大鼠。给幼年ＳＨＲ皮下注射６－ｈｙｄｒｏｘｙｄｏｐａｍｉｎｅ损毁交感神经阻止上述酶活性的改变，同时也阻止血压升高，但二者不相关。损毁成年ＳＨＲ交感神纱影响酶活性，血压也未见显著改变。上述结果提示，在ＳＨＲ发育早期阶段，交感神经的作用是ＳＨＲ高血压发生的一个重要原因     

自发性高血压大鼠冠状动脉平滑肌细胞膜电位的研究

目的：观察自发性高血压大鼠（ＳＨＲ）冠状动脉平滑肌细胞的静息膜电位（Ｅｍ ）及其对血管活性物质ＫＣｌ、ＡＣｈ、ＮＥ的反应性,并与Ｗｉｓｔａｒ大鼠进行了比较。方法：采用先进技术细胞内微电极的方法。结果：ＳＨＲ冠状动脉平滑肌细胞静息膜电位稳定,其均值为－４２．４０±２．７０ ｍ Ｖ,比Ｗｉｓｔａｒ大鼠升高２２％ 左右;ＫＣｌ和ＡＣｈ均引起膜电位去极化,且呈剂量依赖式特点;ＮＥ对膜电位无明显影响。结论;　ＳＨＲ的膜电位比Ｗｉｓｔａｒ大鼠较低,对ＫＣｌ和ＡＣｈ 的反应性明显增强     

老年大鼠心肌肌浆网功能的改变

目的 探讨老年大鼠心肌肌浆网（ＳＲ）钙转运的改变及其在心脏收缩功能障碍中的作用。方法 测定老年和成年Ｗｉｓｔａｒ大鼠心功能。取左心室肌组织制备肌浆网膜,采用Ｍｉｌｌｉｐｏｒｅ滤过法测定心肌ＳＲＣａ＾２＋摄取、Ｃａ＾２＋释放和＾３Ｈ－Ｒｙａｎｏｄｉｎｅ受体结合,并测定心肌ＳＲＣａ＾２＋－ＡＴＰ酶活性。结果 与成年组比较,老年组大鼠左室舒张末压（ＬＶＥＤＰ）升高８２％（Ｐ〈０．０５）,左室内压变化速度     

红细胞抗高血压因子对自发性高血压大鼠血管平滑肌细胞游离钙浓度的影响

观察从人红细胞中提取的抗高血压因子（ＡＨＦ）对自发性高血压大鼠（ＳＨＲ）和正常血压ＷＫＹ大鼠培养的主动脉平滑肌细胞胞内游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）的影响。结果ＳＨＲ和ＷＫＹ大鼠静息［Ｃａ２＋］ｉ无显著差别。ＫＣｌ（６０ｍｍｏｌ／Ｌ）对ＳＨＲ的激活显著大于ＷＫＹ大鼠，ＡＨＦ（１０－４ｇ／ｍｌ）可明显抑制由高钾诱导的［Ｃａ２＋］ｉ升高，对ＳＨＲ的抑制程度明显高于ＷＫＹ大鼠；去甲肾上腺素（ＮＥ，０．１ｍｍｏｌ／Ｌ）对两种大鼠的激活程度无显著差异。ＡＨＦ（１０－４ｇ／ｍＬ）也可明显抑制由ＮＥ（０．１ｍｍｏｌ／Ｌ）所诱导的ＶＳＭＣ［Ｃａ２＋］ｉ升高，对两种大鼠的抑制程度无显著差异。结论ＡＨＦ的降压作用可能与抑制细胞内［Ｃａ２＋］ｉ升高有关。     

血液稀释疗法对不同月龄大鼠脑卒后心肌酶活性的影响

应用４～５月龄成年鼠和２４月龄以上老年Ｗｉｓｔａｒ大鼠，分别在外科手术显微镜下采用电灼法造成大鼠一侧大脑中动脉闭塞２４ｈ后进行血清稀释治疗２ｈ，检测心肌酶活性，结果表明：脑卒中后心损伤程度以老年大鼠为重，经血液稀释治疗后，心肌组织Ｎａ＾＋Ｋ－ＡＴＰａｓｅ，Ｃａ＾２＋－ＡＴＰａｓｅ超氧化物歧化酶（ＳＯＤ），丙二醛（ＭＤＡ）和Ｎ－乙酰－β－Ｄ氨基葡萄苷酶ＮＡＧａｓｅ两个年龄组均有一定程度的变化，以成年     

血液稀释疗法对不同月龄大鼠脑卒中后心肌酶活性的影响

应用４～５月龄成年鼠和２４月龄以上老年Ｗｉｓｔａｒ大鼠，分别在外科手术显微镜下采用电灼法造成大鼠一侧大脑中动脉闭塞２４ｈ后进行血液稀释治疗２ｈ，检测心肌酶活性。结果表明：脑卒中后心肌损伤程度以老年大鼠为重，经血液稀释治疗后，心肌组织Ｎａ＋－Ｋ＋－ＡＴＰａｓｅ，Ｃａ２＋－ＡＴＰａｓｅ，超氧化物歧化酶（ＳＯＤ），丙二醛（ＭＤＡ）和Ｎ－乙酰－β－Ｄ－氨基葡萄糖苷酶ＮＡＧａｓｅ两个年龄组均有一定程度的变化，以成年组治疗效果优于老年组大鼠，说明血液稀释对闭塞单侧大脑中动脉所致成年、老年大鼠心肌损伤有一定的治疗作用。     

胰岛素抵抗高血压大鼠血管舒张功能异常的机制

目的探讨胰岛素抵抗高血压（ＩＲＨ）大鼠血管舒张功能的改变及其可能机制。方法采用离体血管功能实验方法,观察大鼠主动脉的舒张功能及其平滑肌钙代谢功能;应用逆转录多聚酶链反应技术检测主动脉平滑肌Ｃａ２＋－ＡＴＰ酶ｍＲＮＡ的表达水平。结果（１）ＩＲＨ大鼠主动脉舒张速度减慢;（２）用无钙ｋｒｅｂｓ液灌流后苯肾上腺素引起的收缩反应显著低于对照大鼠;（３）ＩＲＨ大鼠主动脉平滑肌Ｃａ２＋－ＡＴＰ酶ｍＲＮＡ表达水平显著低于对照大鼠。结论ＩＲＨ大鼠主动脉舒张功能下降与Ｃａ２＋－ＡＴＰ基因酶ｍＲＮＡ水平表达下降引起的平滑肌Ｃａ２＋代谢功能障碍有关。     

高血压大鼠心脏肌凝蛋白重链mRNA表达和卡托普利的作用

目的：观察自发性高血压大鼠（ＳＨＲ）心肌肥厚时心脏α和β－肌凝蛋白重链（α－ＭＨＣ和β－ＭＨＣ）ｍＲＮＡ表达和卡托普利的保护作用。方法：１５周龄雄性ＳＨＲ口服卡托普利（１００ｍｇｋｇ－１／ｄ）１２周（ＣＡＰ组），同样年龄性别的ＳＨＲ（ＳＨＲ组）和Ｗｉｓｔａｒ－ｋｙｏｔｏ大鼠（ＷＫＹ组）不给药，在同样条件下喂养同样时间，通过称重法计算左室重量指数（ＬＶＩ），使用测微技术测量心肌细胞横径（ＴＤＭ），采用Ｎｏｒｔｈｅｒｎ印迹技术检测左室α和β－ＭＨＣｍＲＮＡ水平。结果：（１）ＳＨＲ组ＬＶＩ和ＴＤＭ显著高于ＷＫＹ组；ＣＡＰ组上述指标显著低于ＳＨＲ组，而与ＷＫＹ组无显著差异；（２）ＳＨＲ组α－ＭＨＣｍＲＮＡ表达明显受抑，β－ＭＨＣｍＲＮＡ表达明显增加；卡托普利抑制α－ＭＨＣｍＲＮＡ水平降低，但不影响β－ＭＨＣｍＲＮＡ水平。结论：高血压肥厚心肌ＭＨＣ基因出现了异常表达，卡托普利可以防止这种现象的发生。     

不同龄SHR心肌细胞内Ca~（2＋）及心肌组织NE含量的变化

采用荧光探针结合计算机图像处理技术测定幼年、成年、老年ＳＨＲ心肌单细胞内游离Ｃａ~（２＋）浓度，采用高压液相色谱法测定幼年、成年、老年ＳＨＫ心肌去甲肾上腺素（ＮＥ）含量。结果显示随着年龄增加，ＳＨＲ心肌细胞内游离Ｃａ~（２＋）增加，而心肌组织ＮＥ含量下降。与老年ＷＫＹ大鼠比较，老年ＳＨＲ心肌细胞内Ｃａ（２＋）含量较高（Ｐ＜０．０１），而其心肌组织ＮＥ含量却较少（Ｐ＜０．０５）。本研究提示心肌组织ＮＥ与ＳＨＲ的血压升高及心肌细胞内游离Ｃａ~（２＋）增多无直接关系。＊Ｐ＜０．０１，ａ．ｖｓ．ＳＨＲ２ｍ，ｂ．ｖｓＳＨＲ６ｍ，ｃ．ｖｓ．ＳＨＲ１２ｍ３不同年龄阶段ＳＨＲ心肌组织ＮＥ含量ＳＨＲ的年龄越大，其心肌组织ＮＥ的含量越少，各年龄组之间均有显著差异（Ｐ＜０．０１）。与老年ＷＫＹ大鼠比较．老年ＳＨＲ是心肌组织ＮＥ含量明显降低（Ｐ＜０．０５）（Ｆｉｇ３）。Ｆｉｇ３ＮＥｃｏｎｔｅｎｔｓｉｎｃａｒｄｉａｃｔｉｓｓｕｅｓＩｎＳＨＲｓｏｆｖａｒｉｏｕｓａｇｅｓａｎｄｅｌｄｅｒＷＫＹｒａｔｓ：Ｐ＜０．０５．＊：Ｐ＜０．０１，ａ．ｖｓ．ＳＨＲ２ｍ，ｂ．ｖｓＳＨＲ６ｍ．ｃ．ｖｓＳＨＲ１２ｍ．ＤＩＳＣＵＳＳＩＯＮＦｕｒａ－２能选择性地     

Silicon-Carbon bond cleavage reactions of Ansa tungstenocene compounds: the [Me2Si] bridge as a site for metallocene functionalization

[Me(2)Si(Cp(Me(2)))(2)]W(H)Cl is obtained via reaction of WCl(6) with a mixture of [Me(2)Si(Cp(Me(2)))(2)]Li(2) and NaBH(4), from which the dichloride [Me(2)Si(Cp(Me(2)))(2)]WCl(2) is obtained via treatment with CHCl(3). [Me(2)Si(Cp(Me(2)))(2)]WCl(2) provides a means to access other ansa tungstenocene compounds, such as [Me(2)Si(Cp(Me(2)))(2)]WH(2), [Me(2)Si(Cp(Me(2)))(2)]WMe(2), and [Me(2)Si(Cp(Me(2)))(2)]WCO. Of most interest, the reactions of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl with organolithium reagents do not yield simple ansa tungstenocene derivatives. Specifically, the reactions of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl with MeLi, Bu(n)Li, or PhLi result in the formation of mixed-ring tungstenocene compounds resulting from C-Si cleavage and functionalization of the ansa bridge, namely (Cp(Me(2)))(eta(5),kappa(1)-C(5)H(2)Me(2)SiMe(2)CH(2))WH, (Cp(Me(2)))[eta(5),kappa(1)-C(5)H(2)Me(2)Si(Me)(Bu(n))CH(2)]WH, and (Cp(Me(2)))[eta(5),kappa(1)-C(5)H(2)Me(2)SiMe(2)(C(6)H(4))]WH, respectively. In contrast to the C-Si cleavage achieved by MeLi, Bu(n)Li, and PhLi, the ansa bridge of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl is inert to Bu(t)Li and the product obtained is the fulvene ("tuck-in") complex [Me(2)Si(Cp(Me(2)))(eta(6)-C(5)MeH(2)CH(2))]WH derived from dehydrohalogenation.     

Hydrogen peroxide in the Burkitt's lymphoma cell line Raji provides protection against arsenic trioxide-induced apoptosis via the phosphoinositide-3 kinase signalling pathway

Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.     

Evidence for P(2)-purinoceptors contribution in H(2)O(2)-induced contraction of rat aorta in the absence of endothelium

OBJECTIVE: H(2)O(2) can contract many arteries, however the underlying mechanisms are not fully understood. This study aims to test whether H(2)O(2)-induced vasoconstriction could be functionally attributed to the activation of P(2)-purinoceptors in rat aorta and to explore its possible signaling mechanisms. METHODS: Isometric tension recording of H(2)O(2) and ATP-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to identify their possible common signaling pathways. RESULTS: Both H(2)O(2) and ATP induced transient phasic contractions in a concentration-dependent manner (1-1000 microM). Removal of endothelium potentiated the contractile responses to H(2)O(2) and to ATP. H(2)O(2) (30 microM)-induced phasic contraction could be abolished by catalase (800 U/ml), but not affected by SOD (150 U/ml), DMSO (5 mM) and apyrase (5 U/ml), suggesting no involvement of O(2)(-), hydroxyl free radicals and ATP release. Also, several receptor antagonists including phentolamine, atropine, methysergide and chlorpheniramine (each 3 microM) were without effect on H(2)O(2) (30 microM)-induced phasic contraction, suggesting no involvement of typical neurotransmitter release. However, both H(2)O(2) (30 microM) and ATP (1 mM)-induced phasic contractions not only presented homologous desensitization, but also showed heterogeneous desensitization. Furthermore, the phasic contractions in response to H(2)O(2) (30 microM) or ATP (100 microM) could be inhibited or abolished in a concentration dependent manner by RB-2 and suramin (10-100 microM), two widely used P(2)-purinoceptor antagonists, with only partial inhibition by Evans blue (300 microM), a moderately selective P(2x) receptor blocker, or by alpha-beta-methylene-ATP (100 microM), a selective P(2x) receptor desensitizer. On the other hand, both H(2)O(2) (30 microM) and ATP (100 microM)-induced phasic contractions were also attenuated, to different degree, by inhibitors of several enzymes including PLC, PKC, PLA(2) and cyclooxygenase. Lastly, removal of extracellular Ca(2+) or pretreatment with procaine (10 mM) and dantrolene (30 microM), two putative intracellular Ca(2+) release blockers, or with Ni(2+) (100 microM) and tetrandrine (5 microM), two Ca(2+) channel blockers, all significantly inhibited H(2)O(2) and ATP-induced contractions. However, nifedipine (1 microM), a voltage-dependent L-type Ca(2+) channel blocker, was without effect. CONCLUSIONS: Our results demonstrate that H(2)O(2)-induced phasic contraction of rat aorta involves, at least in part, the activation of P(2)-purinoceptors in the aortic smooth muscle cells     

A multistep kinase-based sertoli cell autocrine-amplifying loop regulates prostaglandins, their receptors, and cytokines

In Sertoli epithelial cells, the IL-1beta induces prostaglandins (PG) PGE(2), PGF(2alpha) and PGI(2) (7-, 11-, and 2-fold, respectively), but not PGD(2), production. Cyclohexamide pretreatment inhibiting protein synthesis prevents IL-1beta increases in PG levels, indicating that induction requires de novo protein synthesis. IL-1beta-regulated PGE(2) and PGF(2alpha) production and cytokine expression require activation of cyclooxygenase-2 (COX-2) and c-Jun NH(2)-terminal kinase, as shown using specific enzyme inhibition. PGE(2) and PGF(2alpha) stimulate expression of IL-1alpha, -1beta, and -6, findings consistent with PG involvement in IL signaling within the seminiferous tubule. PGE(2) and PGF(2alpha) reverse COX-2-mediated inhibition of IL-1beta induction of cytokine expression and PG production. Sertoli PG receptor expression was determined; four known E-prostanoid receptor (EP) subtypes (1-4) and the F-prostanoid and prostacyclin prostanoid receptors were demonstrated using RNA and protein analyses. Pharmacological characterization of Sertoli PG receptors associated with cytokine regulation was ascertained by quantitative real-time RT-PCR analyses. IL-1beta regulates both EP(2) mRNA and protein levels, data consistent with a regulatory feedback loop. Butaprost (EP(2) agonist) and 11-deoxy PGE(1) (EP(2) and EP(4) agonist) treatments show that EP(2) receptor activation stimulates Sertoli cytokine expression. Consistent with EP(2)-cAMP signaling, protein kinase A inhibition blocks both IL-1beta- and PGE(2)-induced cytokines. Together, the data indicate an autocrine-amplifying loop involving IL-1beta-regulated Sertoli function mediated by COX-2-induced PGE(2) and PGF(2alpha) production. PGE(2) activates EP(2) and/or EP(4) receptor(s) and the protein kinase A-cAMP pathway; PGF(2alpha) activates F-prostanoid receptor-protein kinase C signaling. Further identification of the molecular mechanisms subserving these mediators may offer new insights into physiological events as well as proinflammatory-mediated pathogenesis in the testis.     

Effect of inhaled N-acetylcysteine on hydrogen peroxide exhalation in healthy subjects

N-acetylcysteine (NAC) has antioxidant properties and its oral administration decreased H(2)O(2) exhalation in patients with chronic obstructive pulmonary disease. In this study we tested whether inhaled NAC could suppress H(2)O(2) levels in exhaled breath condensate (EBC) of eight healthy subjects that have never smoked (never-smokers). Original NAC solution (ACC vial, 300 mg NAC in 3 ml solvent), NAC-placebo (vehicle), sterile 0.9% NaCl or distilled water were nebulized via the pneumatic De Vilbiss nebulizer once daily every 7 days and H(2)O(2) and thiols exhalation was measured just before, 30 min and 3 h after the end of drug administration. Additional in vitro experiments were performed to evaluate NAC stability during nebulization, reactivity with H(2)O(2) and possible H(2)O(2) generation in aqueous NAC solutions. NAC almost completely abolished H(2)O(2) exhalation 30 min after inhalation (0.02+/-0.04 vs. 0.21+/-0.09 microM, p<0.001). However, 3 h later the H(2)O(2) levels raised 1.8-fold from baseline (p<0.01). Other inhaled solutions did not affect H(2)O(2) levels. Mean thiol concentration in EBC rose (p<0.05) after treatment with NAC and reached 1.03+/-0.48 microM at 3 h. Although, 25 and 50 mM NAC completely inhibited H(2)O(2)-peroxidase-luminol-dependent chemiluminescence, detectable amounts of H(2)O(2) were generated in NAC solutions. It was accompanied by moderate loss of -SH groups. Catalase and ascorbic acid prevented H(2)O(2) formation in NAC solutions. In conclusion inhaled NAC revealed biphasic effect on H(2)O(2) exhalation in healthy subjects, which depends on direct H(2)O(2) scavenging and H(2)O(2) generation related to drug oxidation. The net result of these processes may determine anti- or pro-oxidant action of inhaled NAC.     

Antagonistic effects of 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 on L-type Ca2+ channels and Na+/Ca2+ exchange in enterocytes from Atlantic cod (Gadus morhua)

There is mounting evidence that vitamin D and its metabolites play important roles in regulating plasma calcium concentrations in teleost fish as in other vertebrates. The aims of the present study were to elucidate the possible cellular target mechanisms for the rapid actions of 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in Atlantic cod enterocytes at physiological doses, and to establish the concentration and thus the physiological range of circulating 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in the Atlantic cod. The plasma concentrations of 25(OH)D(3), 1,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) were 15.3 +/- 2.7nM, 125.1 +/- 12.3pM and 10.1 +/- 23.5nM respectively. Exposure of enterocytes to 10mM calcium (Ca(2+)) evoked an increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)). This increase was suppressed by 24R,25(OH)(2)D(3) dose-dependently, with an EC(50) of 4.9nM and a maximal inhibition of 60%. 24R,25(OH)(2)D(3) (20nM) abolished an increase in [Ca(2+)](i) (approximately 252%) in the control enterocytes exposed to 10microM S(-)-BAYK-8644, suggesting that the hormone acts by inhibiting Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Administration of 20nM 24R,25(OH)(2)D(3) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 20%, indicating a release of Ca(2+) from intracellular stores. Administration of 25(OH)D(3) (20nM) resulted in a biphasic change in the enterocyte [Ca(2+)](i): within 1--5s, it decreased to 87 +/- 12nM below its mean basal [Ca(2+)](i) (334 +/- 13nM), followed by a rapid recovery of [Ca(2+)](i) to a new level, 10% lower than the initial [Ca(2+)](i). The rapid decrease, the recovery rate and the final [Ca(2+)](i) were all affected dose-dependently by 25(OH)D(3), with EC(50) values of 8.5, 17.0 and 18.9nM respectively. Furthermore, the effects of 25(OH)D(3) were sensitive to sodium (Na(+)), bepridil (10microM) and nifedipine (5 microM), suggesting that 25(OH)D(3) regulates the activity of both basolateral membrane-associated Na(+)/Ca(2+) exchangers and brush border membrane-associated L-type Ca(2+) channels. Administration of 25(OH)D(3) (10nM) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 18%, indicating a release of Ca(2+) from intracellular stores. 1,25(OH)(2)D(3) also affected enterocyte [Ca(2+)](i) in a biphasic manner: the rapid decrease, the recovery rate, and the mean final [Ca(2+)](i) were all affected dose-dependently, with EC(50) values of 8.3, 24.5 and 7.7nM respectively. The high EC(50) values for 1,25(OH)(2)D(3) compared with circulating concentrations of 1,25(OH)(2)D(3) (130pM) suggest that this effect is pharmacological, rather than of physiological relevance in enterocyte Ca(2+) homeostasis of the Atlantic cod. It is concluded that 24R,25(OH)(2)D(3) has a physiological role in decreasing intestinal Ca(2+) uptake via inactivation of L-type Ca(2+) channels, whereas the physiological role of 25(OH)D(3) is to increase enterocyte Ca(2+) transport via activation of Na(+)/Ca(2+) exchangers, concurrent with activation of L-type Ca(2+) channels.     

SERUM FREE 1,25-DIHYDROXYVITAMIN D AND THE FREE 1,25-DIHYDROXYVITAMIN D INDEX DURING A LONGITUDINAL STUDY OF HUMAN PREGNANCY AND LACTATION

The changes in three different indices of 1,25-dihydroxyvitamin D (1,25(OH)2D) biological activity were studied longitudinally in 35 women during late pregnancy and lactation and in 26 control women. Measurements were made of maternal serum total 1,25(OH)2D and free 1,25(OH)2D concentration (by centrifugal ultrafiltration) and the free 1,25(OH)2D index (the molar ratio of total 1,25(OH)2D and vitamin D binding protein (DBP]. During late pregnancy total 1,25(OH)2D concentrations were significantly elevated when compared to controls, as were free 1,25(OH)2D and DBP concentrations and the free 1,25(OH)2D index. Serum total 1,25(OH)2D, free 1,25(OH)2D and DBP concentrations all fell dramatically during the first 2 weeks of lactation with total 1,25(OH)2D and free 1,25(OH)2D concentrations falling to levels below those of controls. During the course of lactation both total 1,25(OH)2D and free 1,25(OH)2D levels rose significantly although they were not different from controls at 18 weeks of lactation. In contrast, the free 1,25(OH)2D index fell during the first 2 weeks of lactation, but remained at this level, significantly lower than controls. Neither urinary calcium excretion nor dietary calcium intake correlated with total or free 1,25(OH)2D, DBP, or the free 1,25(OH)2D index. The disagreement in the results of free 1,25(OH)2D concentration and free 1,25(OH)2D index demonstrates that these two approaches to measuring biologically active 1,25(OH)2D are not equivalent. In attempting to account for the increased calcium requirements of human reproduction we conclude that in pregnancy any of the 1,25(OH)2D measurements may be appropriate. In lactation, however, either 1,25(OH)2D is not a major factor or 1,25(OH)2D biological activity is inadequately represented by any of the currently available methods.     

P2Y2 receptor-mediated modulation of estrogen-induced proliferation of breast cancer cells

It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y(2) receptors promote or suppress proliferation in different cancers. In the present study, the methods of methylthiazoltetrazolium (MTT) assay, real-time RT-PCR, Western blot and fluorescent calcium imaging analysis were used to investigate whether P2Y(2) receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y(2) receptors were expressed in both the estrogen receptor alpha (ER(α))-positive breast cancer cell line MCF-7 and the ER(α)-negative breast cancer cell line MDA-MB-231. 17β-Estradiol (17β-E(2)) (1 pM to 1000 nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1 μM) and the ER(α) antagonist methyl-piperidino-pyrazole (MPP, 50 μM), but not by the ER(β) antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50 μM) or ER(β) small interfering RNA. The P2Y(2) and P2Y(4) receptor agonist UTP (10-100 μM) suppressed the viability of breast cancer cells in both MCF-7 and MDA-MB-231 cells. The effect was blocked by suramin (10-100 μM), known to be an effective antagonist against P2Y(2), but not P2Y(4), receptor-mediated responses. 17β-E(2) played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y(2) receptors. 17β-E(2) (0.1-1000 nM) downregulated the expression of P2Y(2) receptors in terms of both mRNA and protein levels in MCF-7 cells. The effect was blocked by ICI 182,780 and MPP, but not PHTPP or ER(β) small interfering RNA. 17β-E(2) did not affect the expression of P2Y(2) receptors in MDA-MB-231. UTP (10-100 μM) led to a sharp increase in intracellular Ca(2+) in MCF-7 cells. Pre-incubation with 17β-E(2) (0.1 μM) attenuated UTP-induced [Ca(2+)](i), which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER(α) receptors, promotes proliferation of breast cancer cells by down-regulating P2Y(2) receptor expression and attenuating P2Y(2)-induced increase of [Ca(2+)](i).