Hales D B, Greene R
Department of Physiology and Biophysics, University of Illinois at Chicago 60612-7342, USA.
Endocrine. 1998 Feb;8(1):19-28. doi: 10.1385/ENDO:8:1:19.
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.
研究了精氨酸加压素（AVP）在睾丸间质细胞中的作用机制，并与佛波醇-13-肉豆蔻酸酯-12-乙酸酯（PMA）和白细胞介素-1β（IL-1β）的作用进行了比较。先前的报道表明，AVP在17α-羟化酶/C17-裂解酶活性水平上抑制睾丸间质细胞睾酮的产生。本研究证实并扩展了这些观察结果，并对比了AVP与IL-1的作用。在所有实验中，从小鼠中分离出巨噬细胞耗尽的睾丸间质细胞，并在开始处理前在原代培养中维持5天。用8-溴-cAMP加递增浓度的AVP或IL-1β处理睾丸间质细胞。AVP导致cAMP刺激的睾酮产生和P450c17 mRNA表达受到显著的剂量依赖性抑制。IL-1β完全抑制cAMP刺激的睾酮产生和P450c17 mRNA表达。PMA是蛋白激酶C（PKC）的已知激活剂，据报道可抑制睾丸间质细胞类固醇生成。睾丸间质细胞表达V1型血管加压素受体，其与PKC激活偶联。IL-1在睾丸间质细胞中的作用机制尚不清楚，但在其他系统中已有人提出PKC途径的激活参与IL-1的作用。因此，将PMA对cAMP刺激的类固醇生成的作用与AVP和IL-1进行了比较。与AVP的作用相似，PMA抑制cAMP刺激的睾酮产生和P450c17 mRNA表达。为了评估PKC可能参与AVP和IL-1在睾丸间质细胞中的作用，测试了PKC抑制剂Calphostin C。10 nM AVP显著抑制cAMP刺激的睾酮产生和P450c17 mRNA表达（p<0.05），而用Calphostin C处理可逆转这种抑制作用。进行了类似的实验以评估PKC在IL-1作用中的作用。与AVP的结果相反，Calphostin C并未逆转IL-对cAMP刺激的P450c17 mRNA表达的抑制作用。为了进一步评估PKC激活，分析了富含肉豆蔻酰化丙氨酸的C激酶底物（MARCKS）的磷酸化。只有AVP和PMA，而不是IL-1β，导致MARCKS磷酸化增加。这些结果证实AVP和PMA激活PKC，并表明IL-1可能不会在睾丸间质细胞中激活PKC。讨论了AVP介导的类固醇生成抑制的意义以及MARCKS磷酸化的潜在作用。
