Sellitti D F, Puggina E, Lagranha C, Doi S Q
Department of Medicine, Endocrinology and Nephrology Divisions, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Endocrinol. 2004 Jan;180(1):23-34. doi: 10.1677/joe.0.1800023.
C-type natriuretic peptide (CNP) and its cognate guanylyl cyclase receptor, the natriuretic peptide receptor B (NPR-B) together constitute a regulatory system that controls cell function via the generation of intracellular cyclic GMP. In this report we have examined the role of cAMP signaling in the regulation of CNP and NPR-B activity in the FRTL-5 rat thyroid follicular cell line. As had been observed earlier with TSH, the cAMP mimetic, dibutyryl cAMP (dbcAMP; 1 mM) induced a significant reduction in CNP-stimulated cGMP generation that was first apparent after 6 h of treatment. The inhibitory effect of dbcAMP on NPR-B was dose dependent, with an EC50 of 0.2 mM. Pretreatment of FRTL-5 cells with either of two protein kinase A (PKA) inhibitors, KT-5720 and H-89, failed to curtail the dbcAMP reduction in NPR-B activity, suggesting that the cAMP pathway leading to inhibition of NPR-B is PKA independent. Whereas either a 30-min or a 24-h treatment with the protein kinase C-activator phorbol myristate acetate failed to alter maximal levels of CNP-stimulated cGMP, a 24-h exposure to the calcium ionophore A23187 reduced CNP-stimulated cGMP to about one-third of control. Pretreatment of FRTL-5 cells with the cell-permeable calcium chelator 1,2 bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, tetraacetoxymethyl ester completely abrogated the cAMP-induced reduction of CNP-stimulated cGMP. Real-time PCR showed no effect of dbcAMP on NPR-B transcript at 3 and 6 h, but indicated a 40% reduction in transcript by dbcAMP at 24 h. In contrast, real-time PCR indicated a 5-fold increase in CNP transcript at 3 h, reaching 15.4-fold above control at 6 h in cells treated with dbcAMP. In addition, immunofluorescence staining of FRTL-5 cells with a specific antibody for CNP-22 showed the presence of cytoplasmic CNP that was up-regulated by incubation with either TSH or dbcAMP. These results suggested that cAMP signaling regulates the natriuretic peptide system in rat thyroid cells by increasing CNP expression, and reducing NPR-B activity. This latter action of cAMP appears to be both PKA independent and calcium dependent, and provides support for a dominant role for calcium in the regulation of NPR-B in the rat thyroid.
C型利钠肽（CNP）及其同源鸟苷酸环化酶受体——利钠肽受体B（NPR - B）共同构成了一个通过生成细胞内环磷酸鸟苷（cGMP）来控制细胞功能的调节系统。在本报告中，我们研究了环磷酸腺苷（cAMP）信号在FRTL - 5大鼠甲状腺滤泡细胞系中对CNP和NPR - B活性调节的作用。正如之前用促甲状腺激素（TSH）观察到的那样，cAMP模拟物二丁酰环磷腺苷（dbcAMP；1 mM）可诱导CNP刺激的cGMP生成显著减少，这种减少在处理6小时后首次明显出现。dbcAMP对NPR - B的抑制作用呈剂量依赖性，半数有效浓度（EC50）为0.2 mM。用两种蛋白激酶A（PKA）抑制剂KT - 5720和H - 89预处理FRTL - 5细胞，均未能抑制dbcAMP对NPR - B活性的降低，这表明导致NPR - B受抑制的cAMP途径不依赖PKA。虽然用蛋白激酶C激活剂佛波酯肉豆蔻酸酯处理30分钟或24小时均未改变CNP刺激的cGMP的最大水平，但暴露于钙离子载体A23187 24小时可使CNP刺激的cGMP降至对照的约三分之一。用可渗透细胞的钙螯合剂1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N'-四乙酸四乙酰甲酯预处理FRTL - 5细胞，可完全消除cAMP诱导的CNP刺激的cGMP的降低。实时定量聚合酶链反应（Real - time PCR）显示，在3小时和6小时时，dbcAMP对NPR - B转录本无影响，但在24小时时，dbcAMP可使转录本减少40%。相反，实时定量聚合酶链反应表明，在3小时时，dbcAMP处理的细胞中CNP转录本增加了5倍，在6小时时达到比对照高15.4倍。此外，用针对CNP - 22的特异性抗体对FRTL - 5细胞进行免疫荧光染色显示，存在细胞质CNP，其通过与TSH或dbcAMP孵育而上调。这些结果表明，cAMP信号通过增加CNP表达和降低NPR - B活性来调节大鼠甲状腺细胞中的利钠肽系统。cAMP的后一种作用似乎既不依赖PKA也依赖钙，并为钙在大鼠甲状腺中NPR - B调节中的主导作用提供了支持。
