Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor

Abstract

As a cis-acting non-depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC-1 tumour cells. Rat Leydig cells and MLTC-1 cells were treated with CAC (5, 10 and 50 μmol/L) or nAChR agonists (50 μmol/L nicotine or 50 μmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC-1 cells at 5 μmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC-1 cells by up-regulating the expression of Lhcgr and Star. Up-regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up-regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.

Keywords: CHRNA4; MLTC-1 cells; adult Leydig cells; cisatracurium; nAChR; testosterone.

Figure 1

Presence of CHRNA4 on rat adult Leydig cells (ALCs). A, Western blotting band. B, immunohistochemistry of adult rat testis cross‐section. CHRNA4‐ is the CHRNA4‐negative control; CHRNA4+ is the CHRNA4‐positive group; and the brown cells are CHRNA4‐positive ALCs. C, Immunofluorescence. CHRNA4− is the CHRNA4‐negative control; CHRNA4+ is the CHRNA4‐positive ALCs; blue is DAPI; green is CHRNA4; and DAPI/CHRNA4 is merged

Figure 2

Effect of nAChR agonists (nicotine and lobeline) and antagonist (cisatracurium, CAC) on testosterone (T) secretion in rat adult Leydig cells (ALCs) and MLTC‐1 cells. A, Dose‐dependent increase in the Level of T after 12 h of CAC (5‐50 µmol/L) treatment in rat ALCs; (B) in MLTC‐1 cells; (C) CAC antagonizes nicotine action in ALCs; (D) CAC antagonizes lobeline action in MLTC‐1 cells. Mean ± SEM, n = 4; Similar letter designates no significant difference between two groups at P < .05

Figure 3

Cisatracurium (CAC) enhances cAMP levels and basal, LH‐ and cAMP‐stimulated testosterone (T) production in adult Leydig cells (ALCs). A, cAMP levels after 12 h treatment; (B) T levels under basal condition, LH stimulation or 8Br‐cAMP stimulation. All groups in panel were cultured for 3 h. Mean ± SEM, n = 4. Similar letter designates no significant difference between two groups at P < .05

Figure 4

Effect of cisatracurium (CAC) on testosterone synthesis when different substrates are added in T biosynthetic pathway. A, 22R‐hydroxychlesterol; (B) pregnenolone; (C) progesterone; (D) androstenedione. All groups were cultured for 3 h. Mean ± SEM, n = 4. * indicates significant difference when compared to control at P < .05

Figure 5

The effect of cisatracurium (CAC) on up‐regulation of steroidogenesis‐related gene expression in adult Leydig cells (ALCs) and MLTC‐1 cells. Rat ALCs or MLTC‐1 cells were cultured with 0‐50 μmol/L CAC for 12 h. The expression of Leydig cell genes was measured by qPCR and calculated relative to Rps16, the internal control. A, ALC genes; (B) MLTC‐1 genes. Mean ± SEM, n = 5. Asterisks (*, **, ***) designate significant differences from the control at P < .05, 0.01 and 0.001, respectively

Figure 6

The effect of cisatracurium (CAC) on the increase of protein levels in adult Leydig cells (ALCs) and MLTC‐1 cells. Rat ALCs and mouse MLTC‐1 cells were cultured with 0‐50 µmol/L CAC for 12 h. The levels of LHCGR, SCARB1, STAR and HSD3B1 were measured by Western blotting and adjusted relatively to β‐actin (ACTINB), the internal control. A, B, Western blot image and quantitative data, respectively, for ALCs; (C, D) Western blot image and quantitative data, respectively, for MLTC‐1 cells. Mean ± SEM, n = 4. Asterisks (*, **, ***) designate significant differences when compared to control at P < .05, 0.01 and 0.001, respectively

Figure 7

The effect of cisatracurium (CAC) on ERK1/2 phosphorylation. Rat adult Leydig cells (ALCs) were cultured with 0‐50 μmol/L CAC for 12 h. The expression of pERK1/2 and ERK1/2 was measured by Western blot and calculated relatively to ACTINB, the internal control. A, Western blot band; (B) total ERK1/2 levels; (C) pERK1/2 levels; (D) ratio of pERK1/2 to ERK1/2. Mean ± SEM, n = 4. Asterisk (**) designates significant difference from the control at P < .01