Calcium transport by rat colon in vitro
Calcium transport by rat colon in vitro
复制标题
体外大鼠结肠钙转运
DOI：
10.1152/ajplegacy.1969.217.1.121
复制
发表时间：
1969
影响因子：
--
通讯作者：
Harrison He
中科院分区：
文献类型：
--
作者：
Harrison Hc;Harrison He
方法通过用于小肠制备的技术（7）制备大鼠结肠的翻转开放环。该环由回盲部远端的结肠的整个长度组成。用0.85%NaCl溶液彻底冲洗结肠内容物，小心地切下附着的结肠系膜，然后将结肠置于冰冷的NaCl-KCl溶液中，然后外翻。外翻的结肠环填充有0.6ml缓冲液（servitol溶液），并置于含有5 ml相同缓冲液（粘膜溶液）的烧杯中。使用三种缓冲溶液：1）含有145 mEq/升Na的Krebs-Henseleit缓冲液，2）通过用0.3M甘露醇溶液代替0.155 M NaCl而等渗的含有50 mEq/升Na的改性溶液，和3）通过用Tris-碳酸氢盐代替Krebs-Henseleit缓冲液中的NaHCO 3和用0.3 M甘露醇代替NaCl溶液而制备的无Na缓冲液。所有缓冲液含有0.25 mM Ca，用45 Ca标记，约025 PC/ml。将外翻的结肠制备物在Dubnoff代谢振荡器中在95%O2 - 5%CO2的气氛中于37 ℃孵育90分钟，同时持续振荡.在孵育期结束时，将环从浴液中取出，通过浸入三个连续的盐水浴中快速洗涤粘膜表面，去除粘附溶液，并通过将血清排入带有几次空气洗涤的量筒中来收集血清。通过在不锈钢板上测量等分试样并在薄窗盖革计数器中计数，分析血清和粘膜溶液中的45 Ca，通过Cotlove氯化物计分析Cl，通过内标火焰光度计分析Na和K。计算血清和粘膜液中45 Ca的比值，作为粘膜-血清钙转运的指标，并产生浓度差。由于在潜伏期开始和结束时，45 Ca的比活度在粘膜液和血清液中相同，因此通过45 Ca测定来测量两种液体中总钙浓度的比值（7）。在体外外翻结肠制备中，不像外翻的小肠袢，
METHODSEverted open-ended loops of rat colon were prepared by the technique used for small-intestine preparations(7). The loops consisted of the entire length of a colon distal to the ileocecal junction. The colonic contents were thoroughly washed out with 0.85% NaCl solution, the attached mesocolon carefully dissected away, and the colon then placed in ice-cold NaCl-KC1 solution prior to eversion. The everted loop of colon was filled with 0, 6 ml buffer (serosal solution) and placed in a beaker containing 5 ml of the same buffer (mucosal solution). Three buffer solutions were used: 1) Krebs-Henseleit buffer containing 145 mEq/liter Na, 2) a modified solution containing 50 mEq/liter Na made isosmotic by substitution of 0.3 M mannitol solution for 0.155 M NaCl, and 3) a Na-free buffer prepared by substitution of Tris-bicarbonate for NaHC03 in the Krebs-Henseleit buffer and of 0.3 M mannitol for the NaCl solution. All buffers contained 0.25 mM Ca labeled with 45Ca,-025 PC/ml. The everted colon preparation was incubated at 37 C for 90 min with constant shaking in a Dubnoff metabolic shaker in an atmosphere of 95% 02-5% CO2. At the end of the incubation period the loop was removed from the bathing solution, the mucosal surface rapidly washed by immersion in three successive saline baths, blotted free of adherent solution, and the serosal solution harvested by draining into a graduated cylinder with several air washes. The serosal and mucosal solutions were analyzed for 45Ca by measuring aliquots onto stainless steel planchets and counting in a thin-window Geiger counter, for Cl by a Cotlove chloridometer, and for Na and K by an internal-standard flame photometer. The ratios of 45Ca in serosal and mucosal fluid were calculated as the index of mucosal-to-serosal calcium transport with development of a concentration difference. Since the specific activity of 45Ca is the same in the mucosal and serosal fluids both at the beginning and at the end of the incubation period, the ratio of the concentrations of total calcium in the two fluids is measured by the 45Ca determination (7). In the in vitro everted colon preparations, unlike the everted small-intestine loops, there is no net