In this study, the intracellular signaling mechanisms through which TNFalpha increases LDH(A4) activity/expression in primary cultures of porcine testicular Sertoli cells were investigated. Studies were focused on sphingomyelin hydrolysis pathway. Treatment of [(14)C]serine-labeled cells with TNFalpha (15 ng/ml, 0.8 nM) resulted in a transient decrease (approximately 20%) in cellular [(14)C]sphingomyelin and in an increase (approximately 27%) in [(14)C]sphingosine that remained elevated for at least 75 min. In the same experiments, no significant changes were detected in ceramide levels. Exogenous sphingosine stimulated LDH(A4) activity and LDHA expression in a dose-dependent manner (ED(50) = 8 microM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was similar whether Sertoli cells were treated with sphingosine (12 microM) or with TNFalpha (20 ng/ml). Although sphingosine mimicked the action of TNFalpha on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory effect represented about 30% of TNFalpha maximal activity. Sphingomyelinase, C2 ceramide, sphingosine 1-phosphate, N, N-dimethylsphingosine, and phosphorylcholine had no significant effect on LDHA expression/LDH(A4) activity. Exogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cells, suggesting its involvement as sphingosine precursor in TNFalpha-stimulated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4) activity stimulated by TNFalpha was decreased by 36.2% by an inhibitor of sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNFalpha effect. Moreover, bisindolylmaleimide (100 nM), a protein kinase C (PKC) inhibitor decreased significantly by 28.7% the TNFalpha effect on LDH(A4) activity but had no effect on the stimulating action of sphingosine, suggesting that if PKC is involved in TNFalpha action, the sphingosine effect on LDH(A4) is unrelated to the PKC activity or inhibition. Together, the present data suggest that in primary Sertoli cell cultures, TNFalpha stimulating action on LDHA expression is partly exerted via sphingomyelin hydrolysis pathway, sphingosine being the active metabolite.