NCI-H295R, a Human Adrenal Cortex-Derived Cell Line, Expresses Purinergic Receptors Linked to Ca2+-Mobilization/Influx and Cortisol Secretion

Abstract

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 (A2A and A2B), P2X (P2X5 and P2X7), and P2Y (P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, and P2Y14) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y1 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 Ca2+-mobilization in the cells, independently of the extracellular Ca2+ concentration. Increases in intracellular Ca2+ 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 Ca2+-mobilization in the presence of extracellular Ca2+ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca2+ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca2+ exposure induced Ca2+-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 P2Y1-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y1 purinergic receptor for intracellular Ca2+-mobilization, and that P2Y1 is linked to SOCE-activation, leading to Ca2+-influx which might be necessary for glucocorticoid secretion.

In situ metabolomics of aldosterone-producing adenomas

“…In fact, considering all genotypes, we were able to demonstrate a positive correlation between tumor size and tumor AMP and ADP content, respectively. Furthermore, purines, including ATP, ADP, and AMP, have been shown to stimulate steroidogenesis in bovine adrenal cells (30) potentially by inhibition of noninactivating K + current, thereby modulating the resting membrane potential (31) and increasing intracellular Ca 2+ levels (32). Therefore, it is prudent to speculate that abundant purines in KCNJ5-mutated APAs could contribute to or modify steroidogenesis as auto-or paracrine signals.…”

Genome‐wide association study for the level of serum electrolytes in Italian Large White pigs

“…A functional P2RY1 is needed for intracellular Ca²⁺ mobilization, a process that is linked to store‐operated calcium entry activation, leading to Ca²⁺ influx, which may be necessary for glucocorticoid secretion (Nishi et al . ). RAP2B is a member of GTP‐binding proteins that are widely upregulated in many types of tumors and promotes migration and invasion of human suprarenal epithelioma.…”

Guanosine triphosphate can directly regulate cortisol production by activating Ca²⁺-messenger systems in bovine adrenal fasciculata cells

“…Moreover, it was reported that NCl-H295R (a human adrenal cortex-derived cell line) expressed multiple purinergic receptors and P2Y1 was related to glucocorticoid secretion [16]. The results of the present study demonstrated that the binding site of GTP seems to be completely different from that of ATP (Fig.…”