Effect of Soluble Adenylyl Cyclase (ADCY10) Inhibitors on the LH-Stimulated cAMP Synthesis in Mltc-1 Leydig Cell Line.

Thi Mong Diep Nguyen,Nguyen Thuy Duong,Laura Filliatreau,Nong Van Hai,Danièle Klett,Yves Combarnous

doi:10.3390/ijms22094641

Abstract

In contrast to all transmembrane adenylyl cyclases except ADCY9, the cytosolic soluble adenylyl cyclase (ADCY10) is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions. In the present paper, we focus on ADCY10 localization and a kinetic analysis of intracellular cAMP accumulation in response to human LH in the absence or presence of four different ADCY10 inhibitors (KH7, LRE1, 2-CE and 4-CE) in MTLC-1 cells. ADCY10 was immuno-detected in the cytoplasm of MLTC-1 cells and all four inhibitors were found to inhibit LH-stimulated cAMP accumulation and progesterone level in MLTC-1 and testosterone level primary Leydig cells. Interestingly, similar inhibitions were also evidenced in mouse testicular Leydig cells. In contrast, the tmAC-specific inhibitors ddAdo3′ and ddAdo5′, even at high concentration, exerted weak or no inhibition on cAMP accumulation, suggesting an important role of ADCY10 relative to tmACs in the MLTC-1 response to LH. The strong synergistic effect of HCO3− under LH stimulation further supports the involvement of ADCY10 in the response to LH.

Highlights

Response to luteinizing hormone (LH) in MLTC-1 cells, we have first checked for its presence by western blot and indirect immunofluorescence, using specific primary antibodies against ADCY10
Western blotting for ADCY10 in MLTC-1 cell revealed a band with an apparent molecular mass of 48 kDa (Figure 1a)
The present study demonstrates the presence of ADCY10 in the MLTC-1 cells and that it plays an important role in the cAMP intracellular accumulation under LH stimulation

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cyclic adenosine 30 ,50 -monophosphate (cAMP) is synthesized from ATP through catalysis by adenylyl cyclases (ADCY) and degraded by catabolizing phosphodiesterases (PDEs). The tmACs reside at the plasma membrane. They are regulated by heterotrimeric G proteins in response to the stimulation of G protein-coupled receptors (GPCRs) by extracellular hormones and neurotransmitters [2]. ADCY10 is found throughout the cytoplasm, and in various organelles, including the nucleus and mitochondria matrix [1,3]. The ADCY10 is insensitive to G proteins and forskolin (a tmAC activator) [4] but is directly activated by calcium and bicarbonate [5,6]

Methods

Results

Discussion

Conclusion