Kodjo M K, Leboulenger F, Conlon J M, Vaudry H
European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, University of Rouen, Mont-Saint-Aignan, France.
Endocrinology. 1995 Oct;136(10):4535-42. doi: 10.1210/endo.136.10.7664674.
It has recently been shown that two novel tachykinins, ranakinin and [Leu3, Ile7]neurokinin A, are present in fibers innervating the frog adrenal gland, and it has been demonstrated that tachykinins stimulate corticosteroid secretion in vitro through activation of chromaffin cells. The purpose of the present study was to investigate the effect of ranakinin on cytosolic free calcium concentrations ([Ca2+]i) and to determine the source of calcium involved. Cultured adrenal cells were loaded with the fluorescent calcium indicator indo-1, and changes in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Administration of a brief pulse of ranakinin (1 microM; 1 sec) in the vicinity of chromaffin cells caused an immediate and transient increase in [Ca2+]i. Repeated pulses of ranakinin resulted in a gradual decline in the [Ca2+]i response, suggesting the occurrence of a desensitization phenomenon. Preincubation of the cells with the calcium channel blockers nifedipine (10 microM) and omega-conotoxin (1 microM) did not alter the response of chromaffin cells to ranakinin. Chelation of extracellular calcium by EGTA (10 mM) caused a marked decrease in the basal [Ca2+]i, but did not suppress the ranakinin-induced [Ca2+]i increase. Conversely, incubation of the cells with thapsigargin (10 microM), an inhibitor of calcium adenosine triphosphatase activity, abolished the stimulatory effect of ranakinin, indicating that the increase in [Ca2+]i can be ascribed to mobilization of calcium from intracellular stores. Preincubation of adrenal cells with the phospholipase C antagonist U-73122 (1 microM; 18 min) or with pertussis toxin (10 microM; 18 h) totally blocked the ranakinin-induced [Ca2+]i rise. Taken together, these data indicate that in frog adrenochromaffin cells, ranakinin causes mobilization of calcium from intracellular stores. The effect of ranakinin is mediated through activation of a phospholipase C via a pertussis toxin-sensitive G protein.
最近研究表明，两种新型速激肽——蛙皮激肽和[亮氨酸3，异亮氨酸7]神经激肽A存在于支配青蛙肾上腺的神经纤维中，并且已经证明速激肽在体外通过激活嗜铬细胞来刺激皮质类固醇分泌。本研究的目的是研究蛙皮激肽对胞质游离钙浓度（[Ca2+]i）的影响，并确定所涉及的钙源。将培养的肾上腺细胞用荧光钙指示剂indo-1负载，并用双发射波长显微荧光测定法研究[Ca2+]i的变化。在嗜铬细胞附近给予短暂的蛙皮激肽脉冲（1微摩尔；1秒）会导致[Ca2+]i立即且短暂地增加。重复给予蛙皮激肽脉冲会导致[Ca2+]i反应逐渐下降，提示发生脱敏现象。用钙通道阻滞剂硝苯地平（10微摩尔）和ω-芋螺毒素（1微摩尔）对细胞进行预孵育不会改变嗜铬细胞对蛙皮激肽的反应。用乙二醇双四乙酸（EGTA，10毫摩尔）螯合细胞外钙会导致基础[Ca2+]i显著降低，但不会抑制蛙皮激肽诱导的[Ca2+]i增加。相反，用毒胡萝卜素（10微摩尔）（一种钙腺苷三磷酸酶活性抑制剂）孵育细胞会消除蛙皮激肽的刺激作用，表明[Ca2+]i的增加可归因于细胞内钙库中钙的释放。用磷脂酶C拮抗剂U-73122（1微摩尔；18分钟）或百日咳毒素（10微摩尔；18小时）对肾上腺细胞进行预孵育会完全阻断蛙皮激肽诱导的[Ca2+]i升高。综上所述，这些数据表明在青蛙肾上腺嗜铬细胞中，蛙皮激肽会导致细胞内钙库中钙的释放。蛙皮激肽的作用是通过一种对百日咳毒素敏感的G蛋白激活磷脂酶C来介导的。
