Possible involvement of ceramide in the regulation of rat Leydig cell function

In the present study, a possible role of a ceramide-dependent pathway in th e regulation of Leydig cell function was investigated. Intracellular cerami de levels were increased by: (a) adding ceramide analogs, (b) inhibiting ce ramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permea ble ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S.2R-D-erythro-2 -(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Shingomyelinase fr om S. aureus lts origin was utilized. Leydig cells were cultured for 3 or 2 4 h with or without the different drugs (10 muM) and SMase (0.3 U/ml) in th e presence or absence of hCG (10 ng,ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-st imulated testosterone production was observed at 3 and 24 h in all cases. T he inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibiti on in hCG-stimulated testosterone production. TNF alpha and IL1 beta. two p ossible inducers of sphingomyelin hydrolysis, produced similar effects on h CG-stimulated testosterone production. In experiments performed in the pres ence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultur es indicating that this pathway inhibits post-cAMP formation events. To stu dy possible loci for the action of ceramide on the steroidogenic pathway, c ells were incubated with C6 and MAPP in the presence of different testoster one precursors. The drugs inhibited testosterone produced from 22(R)-hydrox ycholesterol (22R-OHChol), pregnenolone and 17 alpha -hydroxyprogesterone ( 17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidoge nesis at the level of cAMP production and at post-cAMP events. (C) 2001 Els evier Science Ltd. All rights reserved.