ADP‐sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells

Abstract

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.

Adenosine 5′-(γ-thio) triphosphate (ATPγS) stimulates both P2Y receptors linked to inositol phosphates production and cAMP accumulation in bovine adrenocortical fasciculata cells

“…We have already reported that, while ATP and UTP may bind to P2Y 2 receptor, ADP binds to an unidentified P2Y receptor linked to adenylyl cyclase in BAFC (Nishi et al, 2002). Our findings in the present study indicate that ATPgS binds to both purinoceptors.…”

“…They induce steroidogenesis via the UTP binding sites (e.g. P2Y 2 receptors) (Nishi, 1999) and ADP binding sites (unknown P2Y receptors) (Nishi et al, 2002) in BAFCs. However, the nucleotide potency order on cAMP accumulation and the mRNA level make it unlikely that BAFC has P2Y 1 receptors (Nishi, 1999).…”

“…Furthermore, Nishi had demonstrated the existence of a ADP-sensitive P2Y receptor linked to cyclic AMP (cAMP) accumulation-protein kinase A (PKA), one of the activation systems in BAFCs (Nishi et al, 2002). This P2Y receptor coupled to adenylyl cyclase is still unknown and no other P2Y receptor subtype linked to adenylyl cyclase activation, such as P2Y 11 , has been identified in BAFCs.…”

“…One of the aims of the present study is to identify the effect of ATPgS on BAFCs via P2Y receptors on the cell surface (Nishi et al, 2002). The other is, using the whole cells, to analyze the ATPgS binding sites on the plasma membrane.…”

Mouse Leydig cells express multiple P2X receptor subunits

“…Several studies have shown that extracellular ATP acts as a modulator of function in different endocrine cell types, including Sertoli cells [16], spermatogonia [32], granulosaluteal cells [47], and adrenocortical fasciculate cells [39]. In Leydig cells of rats and mice, ATP leads to an increase in intracellular calcium concentration ([Ca 2+ ] i ) and subsequent testosterone secretion [17,40].…”