ADP-sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells.
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 microM and 40.1+/-5.5 microM, respectively. In contrast, the EC(50) of BzATP for cAMP production was 153.0+/-37.4 microM. Adenosine and AMP (0.1-1000 microM) were much less effective than ADP and ATP. 2MeSADP and UTP did not exert detectable effects. ADP (10 and 100 microM) significantly stimulated steroidogenesis; the process was blocked by an adenylyl cyclase inhibitor SQ22536 (100 microM) but not by the P2Y(1) receptor antagonist MRS2179 (100 microM). 3. Real-time imaging of the PKA activity with the dye ARII, which became less fluorescent upon phosphorylation, revealed that ADP (100 microM) immediately activated PKA. These effects could be mimicked by forskolin (100 microM) and were blocked by the PKA inhibitor H89 (50 microM). UTP (100 microM) did not activate PKA. 4. The cytoplasm harvested from morphologically and electrophysiologically identified single BAFCs contained mRNA for P2Y(2) but not for P2Y(1), P2Y(4), P2Y(11) or P2Y(12) receptors, as confirmed by single-cell RT-PCR amplification (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 purine-activated steroidogenesis via cAMP/PKA-mediated pathways, independently from the pathways involving InsP(3) production and consequent intracellular Ca(2+) increase.
Nishi
Haruhisa
H
Department of Pharmacology, Jikei University School of Medicine, Tokyo 105-8461, Japan. hnishi@jikei.ac.jp
Kato
Fusao
F
Masaki
Eiji
E
Kawamura
Masahiro
M
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000502:physiology*; D000302:Adrenal Cortex / Q000378:metabolism*; D000305:Adrenal Cortex Hormones / Q000096:biosynthesis*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D014544:Uridine Triphosphate / Q000494:pharmacology
D000305:Adrenal Cortex Hormones; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D014544:Uridine Triphosphate