Role of the D2-like dopamine receptors in the regulation of aldosterone secretion of human adrenal cortical cells

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.