Endocrine Abstracts

Introduction: Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past 10 years, important discoveries have been made regarding the genetic basis of aldosterone producing adenoma and familial forms of primary aldosteronism. In most cases, genetic abnormalities are found in genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H, CLCN2) and pumps (ATP1A1, ATP2B3). These mutations affect intracellular ion homeostasis and/or cell membrane potential, leading to increased intracellular calcium concentrations and activation of calcium signalling, which is the main regulator of aldosterone biosynthesis.

Objective: The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA.

Methods: We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α named α7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, steroid production, electrophysiological properties and gene expression.

Results: Treatment of α7-5HT3 expressing cells with increasing concentrations of PSEM-817 (from 10^-9 to 10^-5 M) induced significant cell membrane depolarization, leading to the opening of voltage gated calcium channel and increased intracellular calcium concentrations. Activation of calcium signaling resulted in increased CYP11B2 (coding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8h, 24h, 48h, 72h). Interestingly, calcium kinetics analyses revealed significant differences in response to treatment with PSEM-817, which leads to sodium entry into cells, or with potassium. Gene expression analyses, by RNA sequencing, revealed the activation of different specific signaling pathways in response to treatment with Angiotensin II, potassium and PSEM-817.

Conclusions: Our results suggest that modulation of intracellular sodium concentrations may activate specific signaling pathways, different from those induced by angiotensin II and potassium. This cell line, in which we can modulate the intracellular calcium concentration ‘on demand’ through modulation of sodium entry into the cells, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.