Szafranska B, Tilton J E
Department of Animal and Range Sciences, North Dakota State University, Fargo, 58105-5727, USA.
J Physiol Pharmacol. 2000 Sep;51(3):541-54.
A new experimental model was utilized to study calcium involvement in the mechanism of opioid influence on cultured porcine pituitary cells. The in vitro model involved interactive argon laser cytometry of pituitary cells pre-loaded by three dyes (fluo-3AM, fura-red and naloxone-conjugated to fluorescein). We compared: 1) the kinetics of free intracellular calcium ([Ca2+]i) in anterior pituitary cells of pregnant pigs (day 25-30) treated in vitro with naloxone (NAL) or gonadotrophin-releasing hormone (GnRH) and 2) the distribution of the opioid-sensitive cells by image analysis of doubly loaded cells. In experiment 1, the changes in [Ca2+]i of pituitary cells pre-loaded with fluo-3 AM (488(ex)/520(em) nm) in response to NAL (10(-6) M) or to GnRH (10(-8) M) were compared to a control cell group. Repetitive line scans across cells were performed and the fluorescence emission from individually selected cells was measured in a time-dependent manner (in 0.5 seconds intervals during periods of 50 seconds). Analysis of data indicated significant increases of [Ca2+]i in NAL- (P<0.001) and GnRH-treated cells (P<0.05) in comparison to the control group. In experiment 2, the fluorescence intensity of doubly-loaded pituitary cells (fura-red, 488(ex)/605(em) nm, as principal calcium indicator and NAL-conjugated with fluorescein, 488(ex)/520(em) nm, to distinguish opioid-sensitive cells) were measured using dual detector image analysis. We found that only approximately 8% of the entire population of anterior pituitary cells exhibited sensitivity to the opioid antagonist treatment. This paper demonstrates calcium involvement in the opioid action on anterior pituitary cells from pregnant pigs and provides a useful model for studies at the individual pituitary cell level and in time-dependent manner.
利用一种新的实验模型研究钙在阿片类药物对培养的猪垂体细胞影响机制中的作用。该体外模型涉及对预先用三种染料（fluo - 3AM、fura - red和与荧光素偶联的纳洛酮）加载的垂体细胞进行交互式氩激光细胞术。我们比较了：1）体外经纳洛酮（NAL）或促性腺激素释放激素（GnRH）处理的妊娠母猪（第25 - 30天）垂体前叶细胞中游离细胞内钙（[Ca2 + ]i）的动力学，以及2）通过对双重加载细胞的图像分析来确定阿片类药物敏感细胞的分布。在实验1中，将预先用fluo - 3AM（488（激发）/520（发射）nm）加载的垂体细胞对NAL（10（-6）M）或GnRH（10（-8）M）反应时[Ca2 + ]i的变化与对照组细胞进行比较。对细胞进行重复的线扫描，并以时间依赖性方式（在50秒期间每隔0.5秒）测量单个选定细胞的荧光发射。数据分析表明，与对照组相比，NAL处理组（P < 0.001）和GnRH处理组（P < 0.05）的[Ca2 + ]i显著增加。在实验2中，使用双探测器图像分析测量双重加载的垂体细胞（fura - red，488（激发）/605（发射）nm，作为主要钙指示剂；与荧光素偶联的NAL，488（激发）/520（发射）nm，用于区分阿片类药物敏感细胞）的荧光强度。我们发现，垂体前叶细胞的整个群体中只有约8%对阿片类拮抗剂处理表现出敏感性。本文证明了钙参与阿片类药物对妊娠母猪垂体前叶细胞的作用，并为在单个垂体细胞水平及时依赖性方式进行研究提供了一个有用的模型。
