Abstract Body: Background. Cytosolic and mitochondrial Ca²⁺ concentrations are key signals for triggering the secretion of aldosterone in primary aldosteronism (PA), the most common form of secondary hypertension.

Aim: To investigate [Ca²⁺]_i transients and oscillations in human CD56⁺ cells isolated from aldosterone producing adenoma (APA) and normal surrounding adrenocortical (AAC) cells.
Methods. We assessed [Ca²⁺]_i in CD56⁺ APA and APA-adjacent cells (AAC) in basal conditions and after angiotensin II (Ang II) stimulation using Fura-2 dye, live confocal microscopy, and an ad hoc developed custom-made pipeline to analyze [Ca²⁺]_i signals in terms of frequency and peak parameters as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). As the two-pore domain K⁺ TASK- 2 channels are under expressed in APA, we used quinidine (20 μM), 20’ prior to [Ca²⁺]_i imaging acquisition to examine the effect of the pharmacological inhibition of this channel.
Results. Resting [Ca²⁺]_i basal levels were higher in AAC (n=11) (0.75, IQR;0.90-0.56) than in APA (n=11) (0.36 IQR;0.69-0.33), (p < 0.02). We detected spontaneous Ca²⁺ activity, during resting conditions, in both cell types without significant difference in peak parameters. Upon 1 nM Ang II simulation, AAC showed elevated FWHM, amplitude, and AUC peak parameters of first Ca²⁺ transients. However, APA cells exhibited elevated peak parameters of subsequent Ca²⁺ oscillations. Ca² oscillations frequency and degree of oscillating cells were significantly higher in APA cells than in AAC. After 24 hours in culture, both AAC and APA cells developed rosette-like cell structure resembling the “glomeruli” of the intact zona glomerulosa clusters. Functional Statistics and clustering analysis showed synchronous behavior of cells belonging to the same cluster without significant differences between AAC and APA.
In AAC, Task-2 Inhibition with quinidine tended to increase spontaneous activity and altered peak’s parameters but did not influence frequency of oscillation.

Conclusion. Quantitative assessment of [Ca²⁺]_i signals unveiled significant differences in [Ca²⁺]_i dynamics between human AAC and APA cells. These results suggest the concurrent modulation of frequency and amplitude of [Ca²⁺]_I subsequent oscillations as a potential driver of increased rate of aldosterone production.