Swope S L, Schonbrunn A
Department of Pharmacology, Harvard Medical School, Boston, MA 02115.
Biochem J. 1988 Jul 1;253(1):193-202. doi: 10.1042/bj2530193.
Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.
蛙皮素家族肽能有效刺激克隆胰腺细胞系HIT-T15细胞释放胰岛素。对蛙皮素的反应包括在最初30秒内大量分泌爆发，随后分泌速率有较小幅度升高，并持续90分钟。本研究的目的是确定参与这种双相分泌反应的细胞内信使。向细胞中加入100 nM蛙皮素20秒，可使[3H]二酰甘油（DAG）的细胞内积累增加40%，[3H]肌醇单磷酸（InsP）、双磷酸（InsP2）和三磷酸（InsP3）的积累分别增加40%、300%和800%。相比之下，环磷酸腺苷浓度未受影响。在分泌反应之前的2秒就检测到蛙皮素刺激[3H]InsP3的形成，而分泌反应直到5秒才可以测量。此外，蛙皮素刺激[3H]InsP3生成的效力（ED50 = 14 +/- 9 nM）与其刺激胰岛素释放的效力（ED50 = 6 +/- 2 nM）一致。与其对[3H]InsP3形成的作用一致，蛙皮素在20秒内将细胞内游离Ca2+浓度[Ca2+]i从基础值0.28 +/- 0.01 microM升高到峰值1.3 +/- 0.1 microM。细胞外Ca2+的螯合并没有消除对蛙皮素的分泌反应或[Ca2+]i的升高，表明不需要Ca2+内流。虽然Ca2+离子载体离子霉素（100 nM）模拟了对蛙皮素的[Ca2+]i反应，但它并没有刺激分泌。然而，用离子霉素预处理细胞会降低蛙皮素对[Ca2+]i和胰岛素释放的影响，这表明[Ca2+]i的升高在对该肽的分泌反应中起作用。为了确定蛙皮素刺激后产生的DAG的作用，我们研究了蛋白激酶C的另一种激活剂佛波酯1,2-十四酰佛波醇-13-乙酸酯（TPA）的作用。TPA不影响[Ca2+]i，但在2分钟延迟后增加胰岛素分泌。然而，如果离子霉素与TPA同时加入，则会立即观察到分泌增加。这些数据表明，蛙皮素诱导的初始分泌爆发是由InsP3产生的高[Ca2+]i和DAG激活的蛋白激酶C的协同作用引起的。然而，单独激活蛋白激酶C似乎足以产生持续的分泌反应。
