Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors.
Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors.
复制标题
细胞培养对兔结肠平滑肌缓激肽受体的影响。
DOI：
10.1016/0016-5085(92)91719-k
复制
发表时间：
1992
期刊：
影响因子：
29.4
通讯作者：
Tomomasa,T
中科院分区：
文献类型：
--
作者：
Hyman,PE;Diego,A;Ridout,D;Yuan,QX;Ennes,H;Cominelli,F;SnapeJr,WJ;Tomomasa,T
本研究的目的是评估细胞培养对兔结肠肌细胞缓激肽受体的影响。在从远端结肠制备的纵向肌条中，缓激肽刺激剂量依赖性收缩，其为对氨甲酰胆碱的最大反应的62%。在4 ° C下，[3H]缓激肽与来自远端结肠的新鲜肌肉匀浆的结合是时间依赖性的、饱和的，并且与组织浓度线性相关。0.6 nmol/L [3H]缓激肽的特异性结合为总结合的80% ± 2%。在竞争性结合研究中，希尔系数接近统一，表明存在单一类别的受体。结果表明，缓激肽的活性顺序为：[d-Phe 7]缓激肽>[D-Arg 9]，[Leu 8]缓激肽，与B2受体亚类的结果一致。来自纵肌层的结肠肌细胞达到汇合，并在培养12 - 14天后收获用于研究。缓激肽受体具有高亲和力[解离常数（Kd）= 672 pmol/L]，数量为10，217 ± 2567/细胞。为了表明培养的肌细胞上的受体是功能性的，使用Fura 2测量缓激肽对细胞内钙浓度的影响（a）和使用放射免疫测定法测量缓激肽对培养基中前列腺素E2浓度的影响（B）。在盖玻片上生长至汇合的细胞中，预先加入Fura 2，缓激肽在1 nmol/L时刺激阈值反应，在1 μ mol/L时刺激最大反应（细胞内钙浓度从229增加到633 nmol/L）。缓激肽，100 nmol/L，增加前列腺素E2在培养基中的三倍。总之，结肠肌细胞表达功能性缓激肽受体，与毒蕈碱受体不同，缓激肽受体在培养中持续存在。缓激肽似乎是一个合适的激动剂受体介导的细胞内事件在培养的结肠肌细胞的研究。
The aim of this study was to assess the effect of cell culture on the bradykinin receptor of rabbit colon myocytes. In longitudinal muscle strips prepared from distal colon, bradykinin stimulated dose-dependent contraction that was 62% of the maximal response to bethanecol. At 4 °C, [3H]bradykinin binding to fresh muscle homogenates from the distal colon was time dependent, saturable, and linearly related to tissue concentration. Specific binding of 0.6 nmol/L [3H]bradykinin was 80% ± 2% of total binding. In competitive binding studies, Hill coefficients approached unity, suggesting the presence of a single class of receptors. The order of potency was bradykinin > [d-Phe7]bradykinin ≫ desArg9, [Leu8]bradykinin, which is consistent with results of a B2receptor subclass. Colon myocytes from the longitudinal muscle layer achieved confluence and were harvested for studies after 12–14 days in culture. Bradykinin receptors were of high affinity [disassociation constant (Kd) = 672 pmol/L] and numbered 10,217 ± 2567/cell. To show that the receptors on cultured myocytes were functional, the effect of bradykinin was measured (a) on intracellular calcium concentration using Fura 2 and (b) on prostaglandin E2concentration in the culture media using radioimmunoassay. In cells grown to confluence on cover slips and preloaded with Fura 2, bradykinin stimulated the threshold response at 1 nmol/L and maximal response (increased intracellular calcium concentration from 229 to 633 nmol/L) at 1 μmol/L. Bradykinin, 100 nmol/L, increased Prostaglandin E2in the culture media threefold. In summary, colon myocytes express functioning bradykinin receptors, which, unlike muscarinic receptors, persist in culture. Bradykinin appears to be a suitable agonist for studies of receptor-mediated intracellular events in cultured colon myocytes.