Shah G V, Kennedy D, Dockter M E, Crowley W R
Department of Pharmacology, University of Tennessee-Memphis College of Medicine 38163.
Endocrinology. 1990 Aug;127(2):613-20. doi: 10.1210/endo-127-2-613.
Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.
降钙素（CT）及相关肽，如CT基因相关肽和鲑鱼降钙素（sCT）样肽，存在于大鼠神经系统和垂体中，且sCT能显著抑制基础状态下以及促甲状腺激素释放激素（TRH）刺激的垂体前叶（AP）细胞催乳素释放。由于已知TRH诱导的催乳素释放涉及细胞外和细胞内来源的胞质游离Ca2+增加，本研究的目的是测试sCT是否会干扰这种效应。使用荧光Ca2+指示剂Indo-1 AM和流式细胞术监测急性分散的AP细胞中促分泌剂诱导的胞质游离Ca2+（[Ca2+]i）升高。将AP细胞酶解分散成单细胞悬液，并用20 μM Indo-1 AM加载30分钟。在流式细胞仪中以约500个细胞/秒的速率对加载了Indo-1的AP细胞进行200 - 300秒的扫描，并测定每个细胞中与Indo-1结合的Ca2+产生的荧光与游离Indo-1的荧光之比（Indo-1比率），该比率是[Ca2+]i的一个指标。在基础条件下，AP细胞在扫描过程中显示出稳定的Indo-1比率，并且100%的细胞对Ca2+离子载体离子霉素有Indo-1比率增加的反应。约25 - 30%的AP细胞对1 μM脉冲的TRH有Indo-1比率显著增加的反应，表明[Ca2+]i增加，该反应包括两个阶段，初始的快速上升不受细胞外环境中EGTA存在的影响，随后下降到一个持续的第二阶段，该阶段被EGTA完全消除。在正常的细胞外Ca2+环境中，用100 nM sCT预处理几乎完全抑制了对1 μM TRH的反应。在EGTA预处理的AP细胞中，TRH诱导的[Ca2+]i增加最初对EGTA不敏感的阶段也因预先暴露于sCT而被消除。这些结果表明，sCT通过减弱TRH诱导的[Ca2+]i增加来抑制AP细胞中TRH刺激的催乳素释放，这种效应可能是由于抑制了TRH对Ca2+/磷脂信使系统的刺激作用而产生的。
