NCI-H295R, a Human Adrenal Cortex-Derived Cell Line, Expresses Purinergic Receptors Linked to Ca(2+)-Mobilization/Influx and Cortisol Secretion

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A(2A) and A(2B)), P2X (P2X(5) and P2X(7)), and P2Y (P2Y(1), P2Y(2), P2Y(6), P2Y(12), P2Y(13), and P2Y(14)) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y(1) agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1–1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca(2+)-mobilization in the cells, independently of the extracellular Ca(2+) concentration. Increases in intracellular Ca(2+) concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca(2+)-mobilization in the presence of extracellular Ca(2+) (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca(2+) channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca(2+) exposure induced Ca(2+)-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y(1)-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y(1) purinergic receptor for intracellular Ca(2+)-mobilization, and that P2Y(1) is linked to SOCE-activation, leading to Ca(2+)-influx which might be necessary for glucocorticoid secretion.