Galas L, Lamacz M, Garnier M, Roubos E W, Tonon M C, Vaudry H
Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM U 413), University of Rouen, Mont-Saint-Aignan, France.
Endocrinology. 1999 Jul;140(7):3264-72. doi: 10.1210/endo.140.7.6772.
We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.
我们之前已经表明，促甲状腺激素释放激素（TRH）对青蛙垂体中间叶α-促黑素（α-MSH）分泌的刺激作用与通过电压依赖性钙通道的Ca2+内流、磷脂酶C的激活以及细胞内Ca2+储存的动员有关。本研究的目的是调查蛋白激酶C（PKC）、腺苷酸环化酶（AC）、Ca2+/钙调蛋白依赖性蛋白激酶II（CAM KII）、磷脂酶A2和蛋白酪氨酸激酶（PTK）在TRH诱导的α-MSH释放中的作用。用佛波醇12-肉豆蔻酸酯-13-乙酸酯（24小时）孵育青蛙神经中间叶（NILs），这会导致PKC脱敏，或者用PKC抑制剂NPC-15437孵育，可使TRH对α-MSH释放的作用降低约50%。在大多数促黑素细胞中，TRH诱导细胞溶质Ca2+浓度（[Ca2+]i）持续双相增加。用佛波醇12-肉豆蔻酸酯-13-乙酸酯或NPC-15437预孵育可抑制Ca2+反应的平台期。用TRH（10(-6) M；20分钟）孵育NILs对cAMP产生没有影响。此外，AC抑制剂SQ 22,536不影响NILs对TRH的分泌反应。这些数据表明，磷脂酶C/PKC途径而非AC/蛋白激酶A途径参与TRH诱导的α-MSH释放。钙调蛋白抑制剂W-7和CAM KII抑制剂KN-93没有显著降低对TRH的反应。同样，磷脂酶A2抑制剂奎纳克林和7-7'-二乙氨基乙醇（7-7'-DEA）不损害TRH对α-MSH分泌的作用。PTK抑制剂ST638和酪氨酸-A23对TRH诱导的[Ca2+]i增加没有影响，但以剂量依赖性方式抑制TRH诱发的α-MSH释放（ED50分别为1.22x10(-5) M和1.47x10(-5) M）。综上所述，这些数据表明，在青蛙促黑素细胞中，PKC和PTK参与TRH诱导的α-MSH分泌。PKC的激活负责[Ca2+]i增加的持续阶段，而PTK的激活不影响Ca2+动员。
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