Cortisol

An 18 G cannula was inserted into the antecubital vein of each forearm upon arrival to the research facility. Blood glucose was measured every 10 min (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH, USA). Blood sampling for insulin, counter-regulatory hormones (catecholamines, growth hormone (GH), glucagon, cortisol) and metabolites (lactate, non-esterified fatty acids (NEFAs)) was performed 50 min prior to exercise, at 10, 30, 60, and 80 min of exercise, as well as in the recovery phase (120 min after exercise completion). Insulin, GH, and cortisol were measured using commercially available immunoassay kits (insulin: Architect, Abbott, Baar, Switzerland; GH: Immulite, Siemens, Zurich, Switzerland; cortisol: Modular, Roche, Rotkreuz, Switzerland). Glucagon was measured using a double radioimmunoassay (Siemens, Zurich, Switzerland) in ethylenediaminetetraacetic acid (EDTA) plasma mixed with aprotinin, immediately cooled and frozen after separation. NEFA levels were assessed using a kit from Wako Chemicals (Dietikon, Switzerland). Lactate and pH were determined electrochemically using the ABL 835/837 FLEX (Radiometer, Thalwil, Switzerland) analyser. Plasma catecholamines were quantified using ultraperformance liquid chromatography-tandem mass spectrometry (Waters Acquity UPLC/TQD, Manchester, UK) [19 (link)].

The resected bilateral adrenal adenoma tissues (wet weight, 1 g) were washed twice with Dulbecco’s modified eagle medium (DMEM), cut into small pieces, and dispersed by incubation in DMEM containing 2% collagenase I for 20 minutes at 37°C. Primary cell culture of the adrenal adenomas was performed as described previously (18 (link)). On day 4, when the cells grew to 80% confluence, they were treated with verapamil (10 μM, Sigma-Aldrich) and nifedipine (10 μM, Sigma-Aldrich) or with the vehicle for 24 hours. The culture medium was collected for measuring aldosterone and cortisol, and the concentrations of aldosterone (Sinbe Diagnostic, China) and cortisol (Roche Diagnostics GmbH, Germany) in the medium were directly measured via chemiluminescence.