Assessment of Angiotensin Ii-Induced Calcium Signals in Primary Human Adrenocortical Cells

Objective: Primary aldosteronism (PA), a common form of secondary hypertension, is characterized by an increase of cytosolic intracellular Ca2+ concentrations Ca2+i. Our aim is to investigate transients and oscillations of Ca2+i in a human model of isolated CD56+ aldosterone-producing adenoma (APA) cells and normal surrounding adrenocortical (NAC) cells. Design and method: We monitored Ca2+i, in CD56+ NAC and APA cells in basal condition and after Angiotensin II (Ang II) stimulation using Fura-2 dye and live confocal microscopy, and we developed a custom-made pipeline to analyze calcium signals in terms of frequency and peak parameters like amplitude, full width at half maximum (FWHM) and area under the curve (AUC). To investigate the potential role of the two-pore domain K+ TASK- 2 channel in regulating Ca2+ signals, TASK-2 channels of NAC cells were pharmacologically inhibited using Quinidine (20 microM) 20’ prior to Ca2+i imaging acquisition. Results: Resting Ca2+i basal levels in NAC (n=11) were higher (0.76 a.u.+- 0.04) than in APA (n=12) (0.56 a.u.+- 0.05, p= 0.0087). NAC TASK-2 inhibition decreased resting Ca2+i to 0.58 a.u. +- 0.07 (p > 0.05). Spontaneous Ca2+ activity was higher in NAC with significant higher amplitude and AUC parameters compared to APA cells. Quinidine further increased spontaneous activity in NAC while decreased Amplitude and AUC. For both cell types stimulation with 2 doses of Ang II concentrations 10 -8 and 10 -9 mM, higher Ang II doses promoted higher Ca2+ peak parameters, amplitude and AUC, and a significant decrease in events frequency. Comparison between APA and NAC revealed that, regardless of Ang II dose, first Ca2+ transient parameters are intensified in NAC. However, APA showed consistent elevated subsequent Ca2+ oscillations parameters (Amplitude and AUC), frequency, and degree of oscillation compared to NAC cells stimulated with similar Ang II doses. NAC TASK-2 inhibited cells, when stimulated with 10 -9 mM Ang II, displayed only increased subsequent peak parameters. Conclusions: The bias-free approach designed to quantitatively assess Ca2+i signals, revealed significant differences in Ca2+i dynamics of human NAC and APA cells.