Shah G V, Deftos L J, Crowley W R
Department of Urologic Surgery, University of Kansas Medical Center, Kansas City 66103.
Endocrinology. 1993 Mar;132(3):1367-72. doi: 10.1210/endo.132.3.8440192.
Calcitonin (CT) is a potent and specific inhibitor of basal and TRH-induced PRL release and PRL mRNA levels in rat anterior pituitary (AP) cells, an action mediated through specific inhibition of the Ca(2+)-inositol phosphate messenger system. Because CT and CT-like peptides have been reported to be present in the AP, the present studies investigated 1) whether 35S-labeled CT-like substances can be precipitated from rat AP cell lysates, 2) whether immunoreactive CT is secreted from rat AP cells, as assessed from a cell blot assay and from RIA of spent medium from cultured AP cells, and 3) whether the release of PRL from cultured rat AP cells can be influenced by immunoneutralization of endogenous CT. Dispersed rat AP cells were labeled with [35S]cysteine, CT-like substances were immunoprecipitated from the lysate with antihuman CT (anti-hCT) and antisalmon CT (anti-sCT) immunoglobulin G conjugated to protein-A-Sepharose beads, and the immunoprecipitates were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by fluorography. The results revealed that both anti-sCT and anti-hCT sera precipitated three major bands of 35S-labeled material from AP cell lysates, with approximate molecular masses of 23, 6.5, and 3.5 kilodaltons. Synthetic sCT completely displaced anti-sCT-precipitable bands and partially displaced anti-hCT precipitates. The cell blot assay revealed the presence of CT-immunopositive AP cells, and secretion of this substance was indicated by zones of secretion surrounding these cells. Immunoreactive CT was also detected in spent medium from cultured rat AP cells, and the average rate of release over 4 days of culture was approximately 1 ng sCT eq/3 million AP cells.24 h. Both anti-sCT and anti-hCT sera significantly stimulated PRL release from rat AP cells, and the stimulatory effect of anti-sCT serum was dose dependent up to a concentration of 1:50. The present findings demonstrate that CT-like peptide(s) is synthesized and released from cultured AP cells and suggest that this peptide may participate in the regulation of PRL secretion via paracrine or autocrine actions.
降钙素（CT）是大鼠垂体前叶（AP）细胞基础及促甲状腺激素释放激素（TRH）诱导的催乳素（PRL）释放和PRL mRNA水平的一种强效特异性抑制剂，该作用是通过特异性抑制Ca(2+) - 肌醇磷酸信使系统介导的。由于已有报道称AP中存在CT及CT样肽，因此本研究调查了：1）能否从大鼠AP细胞裂解物中沉淀出35S标记的CT样物质；2）通过细胞印迹分析及培养的AP细胞的培养液放射免疫分析（RIA）评估大鼠AP细胞是否分泌免疫反应性CT；3）内源性CT的免疫中和是否会影响培养的大鼠AP细胞中PRL的释放。将分散的大鼠AP细胞用[35S]半胱氨酸标记，用与蛋白A - 琼脂糖珠偶联的抗人CT（抗hCT）和抗鲑鱼CT（抗sCT）免疫球蛋白G从裂解物中免疫沉淀CT样物质，免疫沉淀物在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上进行分离并用荧光自显影法鉴定。结果显示，抗sCT和抗hCT血清均从AP细胞裂解物中沉淀出三条主要的35S标记物质条带，其近似分子量分别为23、6.5和3.5千道尔顿。合成的sCT完全取代了抗sCT可沉淀条带，并部分取代了抗hCT沉淀物。细胞印迹分析显示存在CT免疫阳性AP细胞，这些细胞周围的分泌区域表明该物质有分泌。在培养的大鼠AP细胞的培养液中也检测到了免疫反应性CT，培养4天的平均释放率约为1 ng sCT当量/300万个AP细胞·24小时。抗sCT和抗hCT血清均显著刺激大鼠AP细胞释放PRL，抗sCT血清的刺激作用在浓度高达1:50时呈剂量依赖性。本研究结果表明，CT样肽在培养的AP细胞中合成并释放，提示该肽可能通过旁分泌或自分泌作用参与PRL分泌的调节。
