At the time of killing, most of the adrenal glands were collected fresh for preparation of a purified, isolated glomerulosa cell preparation as previously reported by us (Braley et al. 1981 (link), 1996 (link), Baudrand et al. 2015 (link), Garza et al. 2015 (link), Chong et al. 2017 (link)). In brief, adrenals were bisected and by blunt scraping the zona glomerulosa (ZG) capsular layer was separated from the fasciculata/medulla. The capsules were suspended in Krebs Ringer bicarbonate solution (Sigma-Aldrich) (0.1% BSA, 200 mg glucose/dl, L-glutamine, 3.7 mmol/L of K+) (KRBGA) solution with collagenase (3.7 mg/mL) and DNAase (0.05 mg/mL) (Worthington Biochemical, Freehold, NJ, USA) for 60-min incubation at 37°C under 95% O2 and 5% CO2. Isolated ZG cells underwent three rounds of brief washing and centrifugation followed by determination of cell count. Purity of the preparation was determined as previously described (Braley et al. 1981 (link), 1996 (link)).
Equal amounts of cells (~50,000) were incubated with 10−7 M angiotensin II (ANGII) or 8.7 mM potassium (K+) for 60 min at 37°C under 95% O2 and 5% CO2. Basal as well as ANGII and K+-stimulated aldosterone secretion levels were determined, and the aldosterone levels normalized to 1 million cells.
Equal amounts of cells (~50,000) were incubated with 10−7 M angiotensin II (ANGII) or 8.7 mM potassium (K+) for 60 min at 37°C under 95% O2 and 5% CO2. Basal as well as ANGII and K+-stimulated aldosterone secretion levels were determined, and the aldosterone levels normalized to 1 million cells.