石杉碱甲对大鼠大脑皮层NMDA受体的调制作用

AIM: To investigate the effects of huperzine A (Hup A) on NMDA receptors in rat cerebral cortex. METHODS: 1) The effect of hup A on NMDA-induced current was studied in acutely dissociated rat hippocampal pyramidal neurons using whole-cell recording. 2) The effect of Hup A on NMDA receptor binding was assessed using [3H] dizocilpine (Diz) binding assay in synaptic membrane preparation of rat cerebral cortex. RESULTS: 1) Hup A reversibly inhibited NMDA-induced current in a concentration-dependent manner with IC50 of 45.4 mumol.L-1. 2) Hup A inhibited the specific binding of [3H]MK-801 to extensively washed synaptic membrane of rat cerebral cortex in a concentration-dependent manner with IC50 of 0.5 (0.1-1.9) mumol.L-1 (n = 4). 3) L-Glutamate 10 mumol.L-1 markedly increased [3H] MK-801 binding. In the presence of L-glutamate, Hup A 0.001-0.1 mumol.L-1 caused a further increase of the binding, whereas Hup A 1-300 mumol.L-1 inhibited the binding in a concentration-dependent manner with IC50 of 12.3 (5.8-26.3) mumol.L-1 (n = 5). CONCLUSION: Hup A acted as an antagonist of NMDA receptor in cerebral cortex in addition to its inhibitory effect on acetylcholinesterase.     

缓激肽B2受体拮抗剂艾替班特降低卡托普和对培养新生大鼠心肌细胞 …

AIM: To study whether endogenous kinins are negative modulators in the growth of cardiomyocytes and their possible cellular and molecular mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were used. Intracellular RNA and protein synthesis were measured by [3H]uridine incorporation and [3H]leucine incorporation, respectively. The expression level of proto-oncogene c-myc, c-fos mRNA was observed by Northern blotting. RESULTS: Exposure of cardiomyocytes to captopril (Cap, 100 mumol.L-1) for 48 h inhibited the rates of [3H]Urd and [3H]Leu incorporations by 25% and 26%, respectively, and for 2 h inhibited c-myc, c-fos mRNA expression by 75% and 55%, respectively. Treatment of angiotensin II (Ang II, 1 mumol.L-1) for 48 h significantly increased the rates of [3H]Urd and [3H]Leu incorporations and for 1 h induced c-myc, c-fos mRNA overexpression, which were reduced by pretreatment with Cap (100 mumol.L-1). Icatibant acetate (Hoe 140, a specific antagonist of bradykinin B2 receptor) 0.1-10 mumol.L-1 blocked the effects of Cap in a concentration-dependent manner. CONCLUSION: Endogenous kinins exhibited a negative modulatory effect on growth of cardiomyoctes via BK B2 receptor.     

丝裂素活化的蛋白激酶反义寡核苷酸抑制表皮生长因子诱导体外 …

目的：探讨丝裂素活化的蛋白激酶（ＮＡＰＫ）反义寡核苷（ＯＤＮ）对表皮生长因子（ＥＧＦ）诱导的培养大鼠血管平滑肌细胞增生的抑制作用。方法：用脂质体将Ｐ４２－和Ｐ４４－ＭＡＰＫ ＯＤＮ０．２μｍｏｌˉＬ＾－１转染入大鼠血管平滑肌细胞，设正义及随机ＯＤＮ为对照，用Ｗｅｓｔｅｒｎ Ｂｌｏｔ法结合Ｐ－８１滤纸法以髓磷脂碱性蛋白为底物测定ＭＡＰＫ活性。［＾３Ｈ］胸腺嘧啶核苷酸掺入测定平滑肌细胞ＤＮＡ合成。结果     

Binding of [3H]-nitrendipine and [3H]-diltiazem to rat myocardial sarcolemma

The binding properties of two major and chemically distinct calcium antagonists, [3H]-nitrendipine and [3H]-diltiazem, were investigated in highly purified rat cardiac sarcolemma. In the case of [3H]-nitrendipine, there appeared a single set of high affinity binding sites with a B max of approximately 0.9 pmol X mg-1 protein and a KD of approximately 0.15 nmol/l. Because of the extremely high value obtained for KD (29 mumol/l), the specificity of [3H]-diltiazem binding seemed questionable. The specific binding of 0.1 nmol/l [3H]-nitrendipine to cardiac sarcolemma was inhibited by nitrendipine and nifedipine (1 mumol/l), only partly inhibited by verapamil (1 mumol/l), and was enhanced by diltiazem (0.1-10 mumol/l). The stimulation of [3H]-nitrendipine binding by diltiazem was associated with an increase in the number of binding sites, Bmax, but with no change in the KD or the Hill coefficient. An enantiomer of diltiazem (1-cis) neither stimulated nor inhibited the [3H]-nitrendipine binding. These results strongly suggest that major prototype calcium antagonists have distinct and variously interacting sites of action in the rat cardiac sarcolemma.     

Leukotriene C4 receptors in cultured smooth muscle cells from bovine anterior cerebral arteries and microcerebrovasculatures.

Specific receptors for leukotriene C4 (LTC4) were identified on smooth muscle cells isolated from bovine anterior cerebral arteries (BACASMC) and bovine microcerebrovasculatures (BMSMC). [3H]LTC4 specific bindings to both cells at a fixed input reached the maxima at 60 min and 20 min, respectively. With incremental inputs of radioligand and a constant cell number, [3H]LTC4 specific bindings reached a plateau indicative of a saturable binding site. Analysis of Scatchard plots demonstrated a single population of high-affinity binding sites in both cells. The dissociation constant (Kd) for BACASMC was 39.2 +/- 1.3 nmol.L-1 and its Bmax was 19.3 +/- 2.1 pmol/10(6) cells. For BMSMC, Kd = 2.0 +/- 0.4 nmol.L-1, Bmax = 157 +/- 13 fmol/10(6) cells. The specific [3H]LTC4 bindings was inhibited by unlabeled LTC4, LTD4 and FPL-55712 (an SRS-A antagonist). The inhibitory rates for BACASMC were 70.4% and 35.3% by LTC4 and FPL-55712 at 1 mumol.L-1, respectively. For BMSMC the inhibitory rates were 96.9%, 73.9%, and 44.9% by LTC4, LTD4, and FPL-55712 at 10 mumol.L-1, respectively.     

小鼠脾脏淋巴细胞上存在μ阿片受体的证据

AIM: Using ohmefentanyl (Ohm), a high affinity mu opioid receptor agonist, to find whether mu opioid receptor exists on mouse spleen lymphocytes. METHODS: The proliferation rates of T-lymphocytes and B-lymphocytes were determined under various concentrations of Ohm with or without naloxone(Nal) in vitro. Binding characteristics of [3H] Ohm with mice spleen lymphocytes were studied by radioligand assays. ReSULTS: Ohm 0.1 pmol.L(-1)-1 nmol.L-1 enhanced Con A-induced spleen T-cell proliferation in vitro. Nal 50 mumol.L-1, which per se enhanced the T-cell proliferation, blocked the enhancing effects of Ohm. However, Ohm had no effect on B-cell proliferation. Furthermore, a satisfactory saturable, specific, and reversible binding was demonstrated with Kd of (6.9 +/- 0.6) nmol.L-1, Bmax of (74 +/- 6) fmol/10(7) cells. The binding of [3H] Ohm was blocked by unlabeled Ohm or Nal. CONCLUSION: Stimulating effects of Ohm on lymphocytes were mediated by opioid receptors. Mouse spleen lymphocytes present mu opioid receptors.     

丝裂素活化蛋白激酶反义寡核苷酸对血管紧张素Ⅱ诱导的大鼠心肌？…

AIM: To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac fibroblast proliferation. METHODS: Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphorothioate-protected 17-mer antisense MAPK oligodeoxynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin II (Ang II) 10 nmol.L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [gamma-32P]ATP and myelin basic protein as substrates. c-myc mRNA expression stimulated by Ang II for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang II for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively. RESULTS: Antisense ODN 0.2 mumol.L-1 reduced Ang II-induced MAPK activities by 72%, MAPK protein expression by 80%, and suppressed c-myc mRNA expression by 97%, respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang II-induced cardiac fibroblast were inhibited by 59% and 58%, respectively. CONCLUSION: A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang II-stimulated cultured neonatal rat cardiac fibroblast proliferation through down-regulating MAPK activity and further depleting c-myc mRNA expression.