Both extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) induced corticoid production (steroidogenesis) concentration-dependently in bovine adrenocortical cells (BA cells). Pertussis toxin (PTX, approx. 2 microg/ml) partially inhibited (approx. 55% inhibition) extracellular ATP (100 microM)-induced steroidogenesis in BA cells. However, PTX did not inhibit extracellular UTP (100 microM)-induced steroidogenesis. Both ATP- and UTP-induced steroidogeneses were significantly inhibited by suramin (50-200 microM). These effects were inhibited significantly by reactive blue-2 (more than 100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (more than 100 microM). Both nucleotides (1-100 microM) induced inositol phosphates accumulation and intracellular Ca2+ mobilization, but PTX did not inhibit them. The RT-PCR procedure identified only P2Y2-receptor mRNA in BA cells. These results suggest that extracellular ATP induces steroidogenesis via a unique P2 receptor linked to PTX-sensitive guanine nucleotide-binding protein (G-protein), while extracellular UTP induces steroidogenesis via P2 receptor linked to PTX-insensitive G-protein. Thus, it was concluded that at least two different P2Y-like receptors linking to steroidogenesis exist in BA cells.
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.
1 The role of P2Y receptors in the production of cAMP and the activation of protein kinase A (PKA) was studied with respect to the regulation of the steroidogenesis in primary cultures of bovine adrenocortical fasciculata cells (BAFCs). 2 ADP and ATP stimulated cAMP production with EC 50 values of 23.7+6.8 mM and 40.1+5.5 mM, respectively. In contrast, the EC 50 of BzATP for cAMP production was 153.0+37.4 mM. Adenosine and AMP (0.1 ± 1000 mM) were much less e ective than ADP and ATP. 2MeSADP and UTP did not exert detectable e ects. ADP (10 and 100 mM) signi®cantly stimulated steroidogenesis; the process was blocked by an adenylyl cyclase inhibitor SQ22536 (100 mM) but not by the P2Y 1 receptor antagonist MRS2179 (100 mM). 3 Real-time imaging of the PKA activity with the dye ARII, which became less¯uorescent upon phosphorylation, revealed that ADP (100 mM) immediately activated PKA. These e ects could be mimicked by forskolin (100 mM) and were blocked by the PKA inhibitor H89 (50 mM). UTP (100 mM) did not activate PKA. 4 The cytoplasm harvested from morphologically and electrophysiologically identi®ed single BAFCs contained mRNA for P2Y 2 but not for P2Y 1 , P2Y 4 , P2Y 11 or P2Y 12 receptors, as con®rmed by single-cell RT ± PCR ampli®cation (50 cycles). 5 These results suggest an expression of an ADP-sensitive G s -coupled purinoceptor in BAFCs. We propose that this not yet described type of P2Y receptor might mediate the extracellular purineactivated steroidogenesis via cAMP/PKA-mediated pathways, independently from the pathways involving InsP 3 production and consequent intracellular Ca 2+ increase.
ABSTRACT-We examined the effect of extracellular adenosine 5'-triphosphate (ATP) on adrenocorticotropic hormone (ACTH)-and angiotensin II-induced steroidogenesis in bovine adrenocortical fasciculata cells. The low concentration of ATP (5 mM) potentiated ACTH-induced steroidogenesis synergistically. However, the purine derivative did not affect angiotensin II-induced steroidogenesis. Although adenosine (100 mM) (a metabolite of ATP) showed a weak steroidogenic effect, it did not potentiate ACTH-induced steroidogenesis. ATP also enhanced the steroidogenesis by NaF synergistically in bovine adrenocortical cells, but did not potentiate forskolin-and dibutyryl cyclic AMP-induced steroidogenesis. The stimulating effect of ACTH on cyclic AMP production was synergistically accelerated by ATP (5 mM), which has no effect by itself on cyclic AMP formation. These results suggest that extracellular ATP affected the ACTH receptor-adenylyl cyclase coupling processes, and potentiation of steroidogenesis by ACTH ensued in bovine adrenocortical fasciculata cells.Keywords: Adrenal cortex, Adrenocorticotropic hormone, Steroidogenesis, ATP, P2Y receptor Extracellular adenosine 5'-triphosphate (ATP) plays many biological roles in diverse cell functions via its cell membrane receptors (1 -4). Receptors for ATP are classified into two groups: a ligand-gated ion channel P2X receptor family and a G protein-coupled P2Y receptor family (5). We previously reported that extracellular ATP stimulated glucocorticoid production (steroidogenesis) in bovine adrenocortical fasciculata cells (BAFC) via P2Y receptors at micromolar concentrations (6).Recently, the importance of cross-talk between two biological active substances on the functions in several kinds of cells and organs has been mentioned. In the case of extracellular ATP, the nucleotide potentiates the insulin secretion stimulated by acetylcholine in isolated perfused rat pancreas (7), and the cytosolic Ca 2+ response to parathyroid hormone in rat osteoblastic cells (8). In steroidogenic organs, the cross-talk between serotonin and angiotensin II (9), interleukin-6 and adrenocorticotropic hormone (ACTH) (10) in rat adrenal steroidogenesis and angiotensin II and ACTH on cyclic AMP formation in bovine adrenal glomerulosa cells (11, 12) had been reported. From these observations, there might be possible cross-talk between extracellular ATP and ACTH (and/ or another steroidogenic substances) on glucocorticoid production in BAFC. Therefore, we investigated the effect of ATP on ACTH-and angiotensin II-induced steroidogenesis in BAFC. MATERIALS AND METHODS Primary culture of BAFCIsolated bovine adrenocortical fasciculata cells were prepared aseptically using collagenase and deoxyribonuclease I as previously described (13). The isolated cells were cultured in Ham's F-10 medium supplemented with 5% fetal calf serum, 10% newborn calf serum, 2.5% horse serum and antibiotics in a 24-well-type dish (10 -15 10 5 cells / well) in a CO2 incubator (humidified atmosphere of 5% CO 2 in air) at 37°C as repor...
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