inositol-1-4-5-trisphosphate and Adrenal-Cortex-Neoplasms

Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-micro (PKC-micro) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP(3)) and calcium ([Ca(2+)](i)). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-alpha/betaII, -epsilon, and -micro but not PKC-delta, -theta, or -zeta/lambda of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-epsilon phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP(3) and [Ca(2+)](i). Inhibition of PKC-epsilon translocation by the PKC-epsilon-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP(3) and [Ca(2+)](i). We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-epsilon phosphorylation and [Ca(2+)](i) elevation.

Topics: Adrenal Cortex; Adrenal Cortex Neoplasms; Aldosterone; Angiotensin II; Calcium; Cell Line, Tumor; Cells, Cultured; Cytochrome P-450 CYP11B2; Humans; Inositol 1,4,5-Trisphosphate; Phosphorylation; Protein Kinase C-epsilon; Receptors, Dopamine D4; RNA, Messenger; Signal Transduction

The mechanism associated with the overproduction of aldosterone by aldosterone-producing adenomas (APA) is unknown.. The objective of the study was to explore the role of the D2 dopamine receptor (D2R) on aldosterone synthesis and secretion and clarify the clinical importance of this role on aldosterone overproduction in APA.. D2R expression in APA was examined in 24 patients and was much less than that in the nontumorous adrenal cortex. D2R mRNA levels in APA were inversely correlated with CYP11B2 mRNA levels and the patient's plasma aldosterone concentration. Angiotensin II (AII)-stimulated aldosterone secretion and CYP11B2 mRNA expression in human adenocarcinoma cells (H295R) was attenuated by the D2 agonist, bromocriptine (BMC). BMC selectively attenuated AII-induced protein kinase C (PKC)-mu phosphorylation and its translocation to the cell membrane. PKCmu-specific short-hairpin RNA significantly decreased AII-induced CYP11B2 mRNA expression and aldosterone secretion. BMC also attenuated the AII-induced increase in cytoplasmic calcium, partially through an inhibition of cytoplasmic inositol 1,4,5 triphosphate production. Despite similar total PKCmu levels in APA and the nontumorous adrenal cortex, expression of phosphorylated PKCmu in APA was much higher.. This is the first study to demonstrate that the D2R modulated aldosterone secretion and synthesis through a specific attenuation of PKCmu activity, as well as the intracellular calcium level. Down-regulation of the D2R in APA, in turn, increased PKCmu activity and led to overproduction of aldosterone in affected patients. The D2R may thus serve as a potential treatment target for primary aldosteronism.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Adenoma; Aldosterone; Angiotensin II; Calcium; Cell Line, Tumor; Cytochrome P-450 CYP11B2; Cytoplasm; Down-Regulation; Humans; Immunoblotting; Inositol 1,4,5-Trisphosphate; Membrane Proteins; Phosphorylation; Protein Kinase C; Receptors, Dopamine D2; Receptors, Dopamine D4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.

Topics: Adrenal Cortex Neoplasms; Aldosterone; Animals; Atrial Natriuretic Factor; Cattle; Cells, Cultured; Cyclic AMP; Humans; Inositol 1,4,5-Trisphosphate; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Vasoactive Intestinal Peptide; Zona Glomerulosa