克隆β细胞中的Ca2+/钙调蛋白抑制作用及磷脂酶C相关的Ca2+信号传导

Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells.

作者信息

Schöfl C, Mader T, Krämer C, Waring M, Krippeit-Drews P, Prank K, von zur Mühlen A, Drews G, Brabant G

机构信息

Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover, Germany.

出版信息

Endocrinology. 1999 Dec;140(12):5516-23. doi: 10.1210/endo.140.12.7180.

DOI:10.1210/endo.140.12.7180

PMID:10579314

Abstract

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10-30 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.

摘要

神经递质和激素，如精氨酸加压素（AVP）和蛙皮素，通过与与磷脂酶C（PLC）相连的受体结合，在胰岛素分泌型HIT-T15细胞中引发频率调制的重复性Ca2+瞬变。通过使用萘磺酰胺类钙调蛋白拮抗剂W-7和W-13及其脱氯对照类似物W-5和W-12，研究了钙调蛋白（CaM）依赖性机制在PLC相关Ca2+瞬变产生中的作用。W-7（10 - 30 microM）和W-13（30 - 100 microM），但不是W-5（100 microM）和W-12（300 microM），可逆地抑制AVP和蛙皮素诱导的Ca2+瞬变。由于PLC相关Ca2+瞬变的产生需要动员细胞内Ca2+以及通过电压敏感性（VSCC）和非电压敏感性（VICC）Ca2+通道的Ca2+内流，因此进一步研究了W化合物对这些过程的影响。首先，W-7剂量依赖性地减少K+（45 mM）诱导的Ca2+信号（IC50，约25 microM），W-13（100 microM）使K+（45 mM）诱导的[Ca2+]i升高降低约40 - 60%，而W-5（100 microM）和W-12（300 microM）没有作用。此外，W-7（100 microM）使小鼠β细胞中的全细胞Ca2+电流抑制约60%。其次，用W-7（30 microM）预处理细胞（5分钟），而不是W-5（30 microM），在无Ca2+培养基中使激动剂诱导的细胞内Ca2+动员抑制约75%。W-7（30 microM）和W-5（30 microM）均不影响AVP（100 nM）刺激的IP3形成。第三，在维拉帕米（50 microM）存在下，由毒胡萝卜素（2 microM）激活的通过VICC的容量性Ca2+内流被W-7（30 microM）抑制，但不被W-5（30 microM）抑制。由于所有W化合物的作用与其报道的抗钙调蛋白活性很好地对应，可以假定存在特异性抗钙调蛋白作用。因此，Ca2+通过激活CaM依赖性过程可以为HIT-T15细胞中PLC相关Ca2+瞬变的产生提供正反馈。这似乎涉及CaM对细胞内Ca2+动员以及通过VSCC和VICC的Ca2+内流的依赖性调节。