Yagil C, Sladek C D
Department of Neurology, University of Rochester School of Medicine and Dentistry, New York 14642.
Endocrinology. 1990 Sep;127(3):1428-35. doi: 10.1210/endo-127-3-1428.
The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)
通过评估高渗环境对大鼠下丘脑 - 神经垂体系统（HNS）器官培养外植体中加压素（VP）mRNA含量的影响，研究了使用该系统的器官培养外植体来研究调节HNS中VP mRNA含量机制的可行性。在渗透压阶梯式升高和在24小时内等量逐渐升高后，观察到了不同的效果。通过RNA保护分析测定的对照HNS外植体的VP mRNA含量为22±6 pg。在培养的24小时和48小时内，其分别逐渐降至对照值的23%和9%。Northern印迹分析显示，培养36小时的外植体中有一条约700个碱基长的VP mRNA条带。暴露于渗透压阶梯式升高的外植体保持静态培养。对照外植体直接放入等渗培养基（299 mosmol/kg H2O）中。暴露于渗透压阶梯式升高的外植体直接放入高渗培养基（大于304 mosmol/kg H2O）中。培养24小时后，测量基础VP释放量，然后所有外植体再暴露于渗透压急性升高15 mosm/kg H2O的环境中。在等渗条件（299 mosmol/kg H2O）下培养的外植体中观察到最高的基础VP释放量。这些外植体对渗透压的急性升高有显著的VP释放增加。在高渗培养基（大于304 mosmol/kg H2O）中培养的外植体基础VP释放量较低，并且这些外植体对急性高渗脉冲无反应。与在等渗培养基中培养的外植体相比，在高渗培养基中培养24小时或48小时的外植体中VP mRNA含量显著降低（分别为47±10%和57±6%；P<0.01）。各组间垂体后叶的VP含量无显著差异。为实现渗透压的缓慢升高，外植体在单独的培养室中以2.1 ml/h的速度用培养基进行灌流。通过增加灌流培养基中的NaCl浓度实现渗透压的逐渐升高（16 mosmol/kg H2O培养基）。对该刺激的反应是VP释放显著增加，并持续9小时。高渗组的VP mRNA含量是对照外植体的165±19%（P<0.001），但与时间对照外植体相比，垂体后叶中的VP含量无差异。（摘要截于400字）
