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.
Highlights
Extracellular nucleotides and nucleosides play important roles in various vertebrate cells and tissues via purinergic receptors expressed on the cell surfaces
It is unknown whether the primers for human message matched the mRNA in bovine or bovine adrenal zona fasciculata cells (BAFC); the latter have no mRNA for these P2Y receptors
The present findings have shown that 2MeS-ATP, as one of the P2Y1-sensitive agonist, is the most effective purinergic agonist for glucocorticoid secretion
Summary
Extracellular nucleotides and nucleosides play important roles in various vertebrate cells and tissues via purinergic (including pyrimidinergic) receptors expressed on the cell surfaces. In other types of animal cells, especially in bovine adrenal zona fasciculata cells (BAFC), there are many reports of functional P2Y receptors for glucocorticoid secretion. Extracellular ATP [7] and UTP (uridine-59-triphosphate) [8] induce glucocorticoid secretion in BAFC, and the subtype of the purinergic receptor in this cell type was identified as P2Y2 [9]. This dominance of P2 over P1 for steroidogenesis is different from that seen in rats. The P2 receptors expressed on adrenocortical cells may have something to do with defense systems under stressful conditions
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