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Objective:Aldosterone producing adenomas (APAs), vary in their clinical presentation, underlying molecular and pathological phenotype. We have investigated the molecular basis of variable aldosterone response to metoclopramide, a dopamine receptor 2 (DRD2) antagonist used to increase the sensitivity of AVS in diagnosing lateralization of aldosterone overproduction.Design and method:We compared the transcriptome of zona glomerulosa (ZG), zona fasciculata (ZF), and APAs in 13 human adrenals, finding 28 genes >5-fold over-expressed in ZG vs ZF. Expression of neurofilament medium (NEFM), a regulator of dopamine receptors, was 14.8-fold higher (p = 9.16E-12) in ZG than ZF, and 4-fold down-regulated in ZF-like APAs carrying KCNJ5 mutations in comparison to small ZG-like APAs (p = −4.35E-03). NEFM protein expression and subcellular localisation were evaluated by immunohistochemistry of human adrenals, and immunofluorescence microscopy in H295R and HEK293 cells. Aldosterone production and secretion and the dopamine receptors gene expression were investigated by silencing NEFM in H295R cells.Aldosterone response to DRD2 antagonist metoclopramide (10–7 M) was measured in H295R cells transfected with mutant DelI157 KCNJ5 constructs.Results:ZG selective expression of NEFM was confirmed by qPCR at 200-fold (cf. ZF). Immunohistochemistry showed ZG selectivity for NEFM staining, which was cytoplasmic and peri-membranous in normal ZG and small ZG-like APAs but absent in the normal ZF and ZF-like APAs. NEFM fluorescence staining in H295R was not filamentous but present in nuclei and cytoplasm. Silencing NEFM in H295R cells down-regulates CYP11B2 and NR4A2 by 40% and 70%, respectively (p = 0.01 and 0.05), and 40% increase of aldosterone secretion after 72 hrs (from 205 to 280 pM/ug protein, p = 0.004). DRD1 and DRD2 were down-regulated by 45% and 70%, respectively (p = 0.007 and 0.02).Aldosterone increase in response to dopamine receptor 2 blockade with metoclopramide was blunted in H295R cells transfected with KCNJ5 mutant constructs whereas it increased by 200% in controls (p = 0.0159).Conclusions:NEFM plays a role in regulating basal aldosterone production and its response to dopamine. Down-regulation of NEFM in ZF-like APAs could explain the low expression levels of DRD2 observed in some APAs, and the variable responses of aldosterone secretion to metoclopramide.

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