Arginine vasopressin inhibition of cytochrome P450c17 and testosterone production in mouse Leydig cells.
The mechanism of arginine vasopressin (AVP) action in Leydig cells was investigated, and compared to the effects of phorbol-13-myristate-12-acetate (PMA) and interleukin-1 beta (IL-1 beta). Previous reports suggested that AVP inhibits Leydig cell testosterone production at the level of 17 alpha-hydroxylase/C17-lyase activity. The present study confirms and extends these observations, and contrasts the effects of AVP to IL-1. In all experiments, macrophage-depleted Leydig cells were isolated from mice and maintained in primary culture for 5 d prior to initiation of treatments. Leydig cells were treated with 8-Br-cAMP plus increasing concentrations of AVP or IL-1 beta. AVP caused a significant and dose-dependent inhibition of cAMP-stimulated testosterone production and P450c17 mRNA expression. IL-1 beta completely inhibited cAMP-stimulated testosterone production and P450c17 mRNA expression. PMA is a known activator of protein kinase C (PKC) and has been reported to inhibit Leydig cell steroidogenesis. Leydig cells express type V1 vasopressin receptors, which are coupled to PKC activation. The mechanism of IL-1 action in Leydig cells is not understood, but activation of the PKC pathway has been suggested for IL-1 action in other systems. Therefore, the effects of PMA on cAMP-stimulated steroidogenesis were compared to AVP and IL-1. Similar to the effects of AVP, PMA inhibited cAMP-stimulated testosterone production and P450c17 mRNA expression. To assess the possible involvement of PKC in AVP and IL-1 action in Leydig cells, the PKC inhibitor Calphostin C was tested. cAMP-stimulated testosterone production and P450c17 mRNA expression were significantly inhibited by 10 nM AVP (p < 0.05), and this inhibition was reversed by treatment with Calphostin C. Analogous experiments were performed to assess the role of PKC in IL-1 action. In contrast to the results for AVP, Calphostin C did not reverse the inhibitory effects of IL-1 on cAMP-stimulated P450c17 mRNA expression. To assess further PKC activation, myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation was analyzed. Only AVP and PMA, but not IL-1 beta, caused an increase in MARCKS phosphorylation. These results confirm that AVP and PMA activate PKC and indicate that IL-1 likely does not activate PKC in Leydig cells. The implications of AVP-mediated inhibition of steroidogenesis and potential role of MARCKS phosphorylation are discussed.
Hales
D B
DB
Department of Physiology and Biophysics, University of Illinois at Chicago 60612-7342, USA. dbhale@uic.edu
Greene
R
R
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000008:administration & dosage / Q000494:pharmacology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007375:Interleukin-1 / Q000008:administration & dosage / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007985:Leydig Cells / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000037:antagonists & inhibitors* / Q000235:genetics; D013739:Testosterone / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D009281:Naphthalenes; D011506:Proteins; D012333:RNA, Messenger; D001127:Arginine Vasopressin; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013739:Testosterone; D013254:Steroid 17-alpha-Hydroxylase; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate