Chang J P, Abele J T, Van Goor F, Wong A O, Neumann C M
Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Gen Comp Endocrinol. 1996 Apr;102(1):88-101. doi: 10.1006/gcen.1996.0050.
In goldfish, growth hormone (GH) release is stimulated by dopamine via D1 receptors and cAMP-dependent mechanisms and by gonadotropin-releasing hormone (GnRH) through a protein kinase C (PKC) pathway; in addition, both D1 and GnRH actions require extracellular Ca2+. In this study, the involvement of arachidonic acid (AA) and calmodulin (CaM) in mediating the GH responses to D1 and GnRH stimulation was examined using primary cultures of dispersed goldfish pituitary cells. In 2-hr static incubation experiments, the phospholipase A2 inhibitor bromophenacylbromide (BPB; 50 microM) decreased the GH responses to the D1 agonist SKF38393 (1 microM), the adenylate cyclase activator forskolin (10 microM), and the cAMP analog 8Br-cAMP (1 mM), but not the responses to salmon (s)GnRH (100 nM), chicken (c)GnRH-II (100 nM), and AA (50 microM). Similarly, the phospholipase A2 inhibitor quinacrine (50 microM) and an inhibitor of AA metabolism, nordihydroguaiaretic acid (NDGA; 50 microM), reduced the GH responses to SKF38393, forskolin, and 8Br-cAMP. The response to the dopamine agonist apomorphine (1 microM) was also decreased by NDGA. The GH responses to AA did not add to those of forskolin or SKF38393, but were additive to responses to sGnRH and the PKC activator tetradecanoyl phorbol acetate (TPA; 100 nM). In perifusion experiments, treatment with BPB reduced the acute GH response to 1 microM SKF38393, 10 microM forskolin, or 1 mM 8Br-cAMP. Taken together, these results suggest that mobilization and metabolism of AA mediate both acute and prolonged GH responses to D1, but not GnRH. The involvement of AA probably occurs distal to D1-induced cAMP generation. Two-hour static incubation with 10 nM to 10 microM KN62, a CaM-dependent kinase II inhibitor, decreased the GH response to 100 nM sGnRH or cGnRH-II. KN62 (1 microM) similarly decreased the GH response to 1 mu M SKF38393, 10 microM forskolin, 1 mM 8Br-cAMP, or 100 nM TPA. In perifusion studies, KN62 (1 microM) also reduced the acute GH response to 5 min pulses of 100 nM sGnRH, 100 nM cGnRH-II, or 1 microM SKF38393. These results indicate that CaM mediates the acute, as well as the prolonged, GH responses to GnRH and dopamine. The involvement of CaM likely occurs distal to cAMP and PKC.
在金鱼中，多巴胺通过D1受体和cAMP依赖性机制刺激生长激素（GH）释放，促性腺激素释放激素（GnRH）则通过蛋白激酶C（PKC）途径刺激GH释放；此外，D1和GnRH的作用均需要细胞外Ca2+。在本研究中，利用分散的金鱼垂体细胞原代培养物，检测了花生四烯酸（AA）和钙调蛋白（CaM）在介导GH对D1和GnRH刺激的反应中的作用。在2小时的静态孵育实验中，磷脂酶A2抑制剂溴苯甲酰溴（BPB；50μM）降低了GH对D1激动剂SKF38393（1μM）、腺苷酸环化酶激活剂福斯可林（10μM）和cAMP类似物8-溴-cAMP（1mM）的反应，但不影响对鲑鱼（s）GnRH（100nM）、鸡（c）GnRH-II（100nM）和AA（50μM）的反应。同样，磷脂酶A2抑制剂奎纳克林（50μM）和AA代谢抑制剂去甲二氢愈创木酸（NDGA；50μM）降低了GH对SKF38393、福斯可林和8-溴-cAMP的反应。NDGA也降低了对多巴胺激动剂阿扑吗啡（1μM）的反应。GH对AA的反应与对福斯可林或SKF38393的反应无叠加，但与对sGnRH和PKC激活剂十四烷酰佛波醇乙酸酯（TPA；100nM）的反应有叠加。在灌流实验中，用BPB处理降低了对1μM SKF38393、10μM福斯可林或1mM 8-溴-cAMP的急性GH反应。综上所述，这些结果表明，AA的动员和代谢介导了GH对D1的急性和长期反应，但不介导对GnRH的反应。AA的参与可能发生在D1诱导的cAMP生成的下游。用10nM至10μM KN62（一种CaM依赖性激酶II抑制剂）进行2小时的静态孵育，降低了GH对100nM sGnRH或cGnRH-II的反应。KN62（1μM）同样降低了GH对1μM SKF38393、10μM福斯可林、1mM 8-溴-cAMP或100nM TPA的反应。在灌流研究中，KN62（1μM）也降低了对100nM sGnRH、100nM cGnRH-II或1μM SKF38393的5分钟脉冲的急性GH反应。这些结果表明，CaM介导了GH对GnRH和多巴胺的急性以及长期反应。CaM的参与可能发生在cAMP和PKC的下游。
