第二類多巴胺受器於人類腎上腺皮質細胞分泌皮質醛酮的調控角色及其機轉的研究

Abstract

此研究的目的是探討第二類多巴胺受器(D2-like dopamine receptors)對腎上腺皮質細胞分泌皮質醛酮(aldosterone)的調控機轉，並藉由針對皮質醛酮分泌性腺瘤(aldosterone producing adenoma, APA)的研究，了解D2-like dopamine receptors在腎上腺高血壓(adrenal hypertension)致病過程中可能扮演的角色。 皮質醛酮於人體是控制鈉鉀離子與體液平衡重要的賀爾蒙，因此可以想見腎上腺分泌皮質醛酮會受到複雜且嚴密的調控。這些調控因子中，血管張力素II (angiotensin II, Ang II)、血中鉀離子濃度、與腦下垂體分泌的腎上腺皮質刺激素(adrenal corticotrophin hormone, ACTH)是最廣為人知的刺激因子，而抑制皮質醛酮分泌的調控因子，則以心房分泌的利鈉激素(atrial natriuretic peptide)以及多巴胺(dopamine)的研究報告最多。血中Ang II濃度可以依體液或鈉離子的不足而改變，因而與調控皮質醛酮分泌以及腎上腺高血壓的形成最有關係。 Dopamine對皮質醛酮的抑制作用在三十年前已有報告，後續一系列的研究發現，單獨給予dopamine並不影響血中皮質醛酮的基礎濃度，但dopamine卻可抑制體液減少或低鈉飲食造成的皮質醛酮濃度上升。另一方面，單獨給予dopamine擷抗劑卻可造成皮質醛酮濃度上升。這些研究結果顯示，dopamine系統在一般生理情形下，扮演一個最大抑制(tonic inhibition)的效果。因此，dopamine系統的缺陷可能會使皮質醛酮的分泌增加，進而造成高血壓的發生。 Dopamine抑制皮質醛酮的作用主要是經由其對D2-like dopamine receptors的作用。我們之前的研究發現，APA的患者對第二類dopamine擷抗劑(metaclopramide)的反應與腺瘤上皮質醛酮合成酶（aldosterone synthase, CYP11B2）的訊息核醣核酸（messenger RNA, mRNA）含量成反比，這暗示著D2-like dopamine receptors的含量（或活性）可能與APA表現CYP11B2的mRNA有關。已知的dopamine receptors共有五種，而腎上腺皮質上只表現D2-like dopamine receptors中的第二型多巴胺受器(D2 dopamine receptor, D2R)與第四型多巴胺受器(D4 dopamine receptor, D4R)，過去我們的研究已發現這兩種dopamine receptors對皮質醛酮分泌有著完全不同的調控效果，在人類腎上腺皮質癌細胞株(NCI-H295R，以下簡稱H295R)上，給予D2R刺激會抑制Ang II刺激皮質醛酮分泌，而給予D4R刺激則會增加Ang II刺激皮質醛酮分泌。 因此，我們首先對手術切除的APA與其同側的無腫瘤部份的腎上腺皮質組織分析Ang II第一型受器（angiotensin II type 1 receptor, AT1R)、D2R與D4R的蛋白質與mRNA的表現量。無論是蛋白質或是mRNA，AT1R在APA與其無腫瘤的腎上腺皮質組織的表現量並無不同，而D2R與D4R的表現量於APA上則明顯減少。但不論是APA或無腫瘤的腎上腺皮質組織，D2R的表現量皆遠較D4R豐富。APA表現CYP11B2的mRNA遠較無腫瘤的腎上腺皮質組織為多百倍以上，且APA表現CYP11B2的mRNA量與患者血中皮質醛酮濃度成正比，而APA之D2R的mRNA表現量則與患者血中皮質醛酮濃度成反比，APA之D2R的mRNA表現量同時也與其CYP11B2的mRNA表現量成反比。 以D2R刺激劑，bromocriptine(BMC)，處理H295R細胞會抑制Ang II增加的皮質醛酮急性及慢性分泌，也會抑制H295R細胞轉錄CYP11B2；若同時給予D2R擷抗劑raclopride(Racl)，則這抑制效果會被逆轉。於H295R細胞轉殖D2R干擾核醣核酸(shRNA)減少D2R表現後，Ang II刺激的皮質醛酮分泌會進一步增加；若加上dopamine，Ang II可刺激此細胞株分泌更多的皮質醛酮；以Racl進一步阻斷少數仍然存在的D2R後，Ang II刺激下的皮質醛酮分泌更進一步增加，對CYP11B2 mRNA的觀察也顯示類似的結果。以特定抗體偵測不同磷酸化的蛋白磷酸酶C (protein kinase C, PKC)亞型，發現Ang II可刺激PKC α/β、μ、ε的磷酸化與轉位至細胞膜，這幾個PKC的活化只有PKC μ會受到BMC的抑制，此抑制作用一樣可用Racl逆轉。於H295R細胞轉殖PKC μ干擾核醣核酸(shRNA)可減少PKC μ及Ang II刺激的PKC μ磷酸化，並導致Ang II刺激的皮質醛酮分泌與CYP11B2 mRNA表現量顯著減少。此外，對照無腫瘤腎上腺皮質組織，表現D2R較少的APA也擁有較多的磷酸化PKC μ。如同之前的報告，Ang II刺激的皮質醛酮分泌與CYP11B2 mRNA表現需inositol 1,4,5 triphosphate (IP3)受器的活化與鈣離子訊息，我們也發現給予BMC可抑制Ang II刺激下細胞內IP3與游離鈣的增加。 關於D4R的研究部分，我們發現於APA、無腫瘤腎上腺皮質組織、培養的正常人類腎上腺皮質細胞與H295R細胞上均有表現D4R。給予D4R刺激劑PD168,077(PD)可加強Ang II刺激的急性與慢性皮質醛酮分泌以及CYP11B2 mRNA表現，而給予D4R擷抗劑L745,870(L)可逆轉此作用。AII刺激PKC α/β、μ、ε磷酸化與轉位至細胞膜，PD選擇性地加強PKC ε 的活化。給予PKC ε的抑制胜肽來抑制磷酸化的PKC ε轉位到細胞膜之後，可顯著減少Ang II刺激的皮質醛酮分泌以及CYP11B2 mRNA的表現。對於鈣離子訊息的調控方面，PD也加強了Ang II刺激下的細胞內IP3與游離鈣增加，同時給予L可逆轉PD的作用。給予細胞內游離鈣的鉗合劑BAPTA並不影響Ang II活化PKC ε；相反地，給予PKC ε的抑制胜肽則顯著影響Ang II刺激後的細胞內IP3與游離鈣增加。Ang II刺激的細胞內游離鈣增加於刺激後幾秒鐘就發生，但D4R刺激劑對Ang II磷酸化PKC ε的影響卻在幾分鐘後才比較明顯。因此，D4R對Ang II刺激的游離鈣增加可直接由影響細胞內IP3的量以及間接藉加強PKC ε的活化而達成。 最後，我們也探討了第二類dopamine受器對AII刺激腎上腺皮質細胞增生的影響。如同之前的認知，AngII可以促進初級培養(primary culture)的人類腎上腺皮質細胞的增生。BMC可抑制Ang II刺激的細胞增生，若再給予Racl，則這種抑制作用會被部分逆轉。BMC並不會增加H295R細胞凋亡(apoptosis)。Ang II可刺激H295R細胞ERK1/2的磷酸化，而BMC則顯著抑制了Ang II磷酸化ERK1/2的作用。分析APA與其無腫瘤的腎上腺皮質組織上磷酸化ERK1/2的量，也發現表現D2R較少的APA擁有較多的磷酸化ERK1/2。D2R對細胞週期(cell cycle)的影響方面， BMC並不會誘發抑癌基因P21、P27或與P53的表現；然而，Ang II刺激H295R的cyclin D1增加則受到BMC的抑制，但表現D2R較少的APA與無腫瘤的腎上腺皮質組織上cyclin D1的表現量並無明顯差異。給予PD98059抑制ERK1/2的磷酸化後，H295R細胞的增生也顯著減少。因此，D2R的表現量減少，可使腎上腺皮質細胞對Ang II刺激的細胞增生作用加強，這可能是腎上腺APA形成的原因之一。 總而言之，我們以一個臨床上較為均質性的腎上腺高血壓次群─APA─為研究對象，發現APA上D2R表現顯著減少，這種D2R減少可能引起PKCμ磷酸化增加而使皮質醛酮分泌增加，我們也藉由細胞培養的實驗證實D2R與D4R這兩種dopamine受器迥異的生理功能，藉著分子生物學的技術，我們觀察到D2R與D4R分別影響Ang II刺激下不同訊息分子的變化，也證實這些被影響的訊息分子對於皮質醛酮分泌與腎上腺皮質細胞增生的角色。同時，我們也於APA觀察到這些訊息分子的變化，因而強化了我們於細胞實驗上的發現之臨床意義。

Previous studies have shown that dopamine inhibited angiotensin II (AII)- or low salt diet-induced increase of plasma aldosterone concentration (PAC) through the D2-like dopamine receptors. Our previous work showed that belong to the D2-like dopamine receptors, both D2 and D4 dopamine receptor (D2R and D4R) expressed on human adrenal cortex and aldosterone producing adenoma (APA) and their physiologic function seemed different. Therefore, my main subject was to explore the role of D2-like dopamine receptors in the regulation of aldosterone secretion of human adrenal cortical cells. By the way of understanding the cell molecular change in APA, we wished to discover the role of D2-like receptors in the pathogenesis of this subtype of human adrenal hypertension. The molecular mechanisms of D2R and D4R were studied by series of experiments. Aldosterone is the most important mineralocorticoid, which regulates sodium and potassium concentration and maintains the adequacy of body fluid. Consequently, the secretion of aldosterone must be under a precise and complicated control. Among the many regulators, AII, plasma potassium concentration, and the adrenal corticotrophin hormone are the most important stimulators. There have been many literatures discussing their role and the regulatory mechanisms of aldosterone secretion. Plasma AII concentration may rapidly respond to body fluid deficiency and salt depletion, therefore it plays the main role in regulation of aldosterone secretion and blood pressure regulation. On the other hands, the inhibitory regulators of aldosterone secretion were much less discussed and far from being understood. Among the inhibitory regulators, atrial natriuretic peptide and dopamine are relatively more reported. The inhibitory role of dopamine in the aldosterone secretion was first reported about 30 years ago. Dopamine did not alter the basal PAC, but it inhibited the increase of the PAC under volume depletion or salt depletion. On the other hand, dopamine antagonist, metoclopramide induced the increase of the PAC. These reports suggested that the dopamine system has a tonic inhibitory effect on aldosterone secretion in the usual physical condition. Dopamine and its antagonist have similar effects on cultured bovine or rat adrenal cortical cells. This finding showed that dopamine inhibited aldosterone secretion could be directly acting on the adrenal cortical cells rather than by the way of indirectly modulating the other regulators involving the aldosterone secretion. The earlier studied have demonstrated that dopamine has its inhibitory effect on aldosterone secretion through D2-like dopamine receptors. Our previous study revealed that the increase of the APA patients’ PAC by metoclopramide was inversely correlated to the expression of CYP11B2 mRNA of these adenomas. This result hinted that the more D2-like dopamine activity, the less CYP11B2 expression in the APA. There are five dopamine receptors discovered. Among the D2-like dopamine receptors, except D3 dopamine receptor, both D2 and D4 dopamine receptors’ mRNA expressed in human adrenal cortex and aldosterone producing adenoma. By different pharmacological inhibitors, we have shown that these two D2-like dopamine receptors seemed to play opposite regulatory roles in aldosterone secretion. In this project, we analyzed the surgical specimen of APA patients to compare the expression of CYP11B2, angiotensin II type 1 receptor, D2R and D4R. We found that the APA had less D2R and D4R than the non-tumor adrenal cortex. The amount of AR1R of the tumor portions was similar to that of the non-tumor adrenal cortex. As expected, the tumor portions had much more CYP11B2 mRNA than the non-tumor adrenal cortex. In consistence with the protein analysis, both D2R and D4R mRNA of the tumor portions were less than those of the non-tumor adrenal cortex, and the mRNA of AT1R of the tumor portions and non-tumor adrenal cortex were similar. By linear regression analysis, we found that the patients’ PAC was positively correlated to the CYP11B2 mRNA expression and negatively correlated to the D2R mRNA expression. On the other hand, the patients’ PAC did not have significant correlation with AT1R and D4R mRNA. The expression of D2R mRNA was more abundance than D4R mRNA in both the tumor portions and the non-tumor adrenal cortex. In order to understanding the cause-result relationship between the D2R decrease and the PAC increase, we used the human adrenal cortical carcinoma cell line, NCI-H295R (H295R), as a cell model to test the role of D2R in regulation of aldosterone secretion. D2R agonist, bromocriptine, did not alter the basal aldosterone secretion. But it significantly inhibited the AII (10-8 mol/L)-stimulated acute (30 min) and chronic (24 hr) aldosterone secretion. Bromocriptine also attenuated the AII-stimulated CYP11B2 mRNA expression. The effect of bromocriptine could be revered by simultaneously giving D2R antagonist, raclopride. In order to mimic the down-regulation of D2R in APA, we used shRNA of D2R to generate a D2R-depleted clone of H295R cells. The D2R-depleted H295R cells have similar basal 24 hr aldosterone secretion and CYP11B2 mRNA expression. Under AII treatment, the D2R-depleted H295R cells have more aldosterone secretion and CYP11B2 mRNA expression than the wild type H295R cells. Dopamine did not alter aldosterone secretion and CYP11B2 mRNA expression in wild type H295R cells. However, dopamine significantly enhanced AII-stimulated aldosterone secretion and CYP11B2 mRNA expression in D2R-depleted H295R cells. Giving the D2R antagonist, raclopride, to block the residual D2R, the enhancing effect of dopamine was further augmented. To understanding the mechanism of the D2R modulation of the aldosterone secretion, we examined the AII-induced PKC and calcium signaling pathway. AII induced phosphorylation of PKC α/β、μ、ε as wells as their translocation to cell membrane. Bromocriptine significantly attenuated AII-stimulated PKCμ (Ser916) phosphorylation and its translocation to membrane. We also observed the reciprocal change of cytoplasmic PKCμ. The effect of bromocriptine on PKCμ activation could be reversed by raclopride. Depleting 60% PKCμ by PKCμ-specific shRNA attenuated AII-stimulated CYP11B2 mRNA expression and aldosterone secretion. We also demonstrated that the APA expressed more abundant phospho-PKCμ than the non-tumor adrenal cortex. In consistence with previous reports, AI-stimulated aldosterone secretion and CYP11B2 mRNA expression were both calcium dependent. Bromocriptine attenuated AII-stimulated increase of cytoplasmic inositol 1,4,5 triphosphate and [Ca2+]。 We demonstrated both D4R and AT1R expression in APA, human normal adrenal cortex, primary cultured human adrenal cortical cells, and H295R cells. AII stimulated aldosterone secretion and CYP11B2 mRNA expression in primary cultured human adrenal cortical cells as well as in H295R cells. But the former responded more to AII stimulation. D4R agonist, PD168,077 enhanced AII-stimulated aldosterone secretion and CYP11B2 mRNA expression in both cultured cells. D4R antagonist, L745,870 reversed the effect of PD168,077. AII stimulated PKC α/β、μ、ε phosphorylation and translocation to cell membrane in primary cultured human adrenal cortical cells as well as in H295R cells. PD168,077 selectively enhanced PKC ε activation. Transferring PKC ε-selective inhibitory peptide to prevent PKCε translocation to cell membrane attenuated AII-stimulated aldosterone secretion and CYP11B2 mRNA expression. PD168,077 also enhanced AII-stimulated increase of cytoplasmic IP3 and [Ca2+]. L745,870 could reverse this effect of PD168,077. Intracellular [Ca2+] chelator, BAPATA, did not inhibit AII-stimulated PKCε phosphorylation. On the other hand, transferring PKC ε-selective inhibitory peptide attenuated AII-stimulated increase of cytoplasmic IP3 and [Ca2+]. AII induced the increase of cytoplasmic [Ca2+] within few seconds, but PD167,077 took several minutes to enhance AII-stimulated PKCε phosphorylation. This result suggested that D4R could augment AII-stimulated cytoplasmic [Ca2+] increase directly by increasing cytoplasmic IP3 or indirectly by enhancing PKCε phosphorylation. Finally, we tried to understand the role of D2R on the tumorigenesis of APA. In consistence with previous reports, AII stimulated proliferation of primary cultured human adrenal cortical cells. Bromocriptine inhibited this cell proliferation, and raclopride reversed it. Bromocriptine did not induce H295R cells apoptosis, but it significantly inhibited the DNA synthesis H295R cells. Bromocriptine attenuated AII-stimulated ERK1/2 phosphorylation and thereafter ERK1/2 translocation from the cytosol to the nuclear in H295R cells. PD98059 which inhibited ERK1/2 phosphorylation also inhibited the proliferation of H295R cells. Analyzing the APA surgical specimen, we found that APA expressed much more phosphorylated ERK1/2 than the non-tumor adrenal cortex, though the total ERK1/2 amounts were similar in APA and the non-tumor adrenal cortex, Bromocriptine did not alter the expression of p21, p27, and p53 of H295R cells, Bromocriptine attenuated AII-stimulated cyclin D1 expression in primary cultured human adrenal cortical cells. Here, we demonstrated the inhibitory effect of D2R on the proliferation of adrenal cortical cells by attenuating the ERK1/2 phosphorylation. Consequently, down-regulation of D2R at least partially contributed to the increase of the ERK1/2 phosphorylation in APA and its tumorigenesis. In conclusion, we focused on APA, a relative homogenous subgroup of the hypertensive patients, to discuss the role of D2-like dopamine receptors in the human adrenal hypertension. The decreased D2R expression in APA negatively correlated to CYP11B2 mRNA expression in APA as well as the patients’ PAC. We further showed the opposite functions of D2R and D4R in the cultured cell models. We demonstrated their effects on the different AII signaling molecules, and the role of these signaling molecules in AII-stimulated aldosterone secretion were proved by the molecular biology techniques. We also provided evidence that D2R inhibited the proliferation of the adrenal cortical cells. Finally, we showed the difference of these signaling molecules between APA and the non-tumor adrenal cortex that confirmed the significance of the signaling molecular modification in the clinical disease, APA.