Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells

Abstract

Involvement of intracellular Ca(2+) and ERK1/2 phosphorylation in the fast nongenomic effects of androgens in myotubes was investigated. Testosterone or nandrolone produced fast (<1 min) and transient increases in intracellular Ca(2+) with an oscillatory pattern. Calcium signals were slightly reduced in Ca(2+)-free medium, but lack of oscillations was evident. Signals were blocked by U-73122 and xestospongin B, inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway. Furthermore, IP(3) increased transiently 2- to 3-fold 45 sec after hormone addition. Cyproterone neither affected the fast Ca(2+) signal nor the increase in IP(3). Calcium increases could also be induced by the impermeant testosterone conjugated to BSA, and the effect of testosterone was abolished in cells incubated with guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. Stimulation of myotubes with testosterone, nandrolone, or testosterone conjugated to BSA increased immunodetectable phosphorylation of ERK1/2 within 5 min, and this effect was not inhibited by cyproterone. Phosphorylation was blocked by the use of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester, U-73122, and xestospongin B as well as by dominant negative Ras, MAPK kinase (MEK), or the MEK inhibitor PD-98059. In addition, guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin blocked ERK1/2 phosphorylation. These results are consistent with a fast effect of testosterone, involving a G protein-linked receptor at the plasma membrane, IP(3)-mediated Ca(2+) signal, and the Ras/MEK/ERK pathway in muscle cells.

Testosterone induction of prostaglandin-endoperoxide synthase 2 expression and prostaglandin F2α production in hamster Leydig cells

“…DHT did not activate MAPK3/1 or PI3K/AKT signalling pathways in hamster Leydig cells (results not shown). Although testosterone failed to activate PI3K/AKT (results not shown), this androgen induced a rapid phosphorylation of MAPK3/1 within short-term incubations (1-3 min), which was also detected after 2-3 h. These results are similar to those observed in studies performed in breast cancer, skeletal muscle cells, prostate stroma cells and Sertoli cells in which androgen activates MAPK3/1 too quickly to be explained by the classical androgen receptor pathway (Peterziel et al 1999, Zhu et al 1999, Estrada et al 2003, Fix et al 2004, Rahman & Christian 2007. We also found that Bi prevented the effects of testosterone on phospho-MAPK3/1 indicating that androgen receptors are required for testosterone-mediated MAPK activation.…”

Androgen regulation of satellite cell function

“…The use of dominant negative mutants of Ras and MEK demonstrated that androgens activate the Ras/MEK/ERK pathway (Estrada et al 2003). The actions described above were not blocked by cyproterone acetate, an inhibitor of the AR, indicating that distinct non-genomic pathways exist for the action of androgens in skeletal muscle cells (Estrada et al 2000(Estrada et al , 2003.…”

“…Although no studies have examined potential nongenomic effects of androgens on myoblasts, non-genomic pathways have been observed in rat myotubes treated with androgens (Estrada et al 2000(Estrada et al , 2003. These effects were characterized by an early increase in intracellular calcium, triggered 15-30 s after testosterone addition, preceded by an increase in inositol 1,4,5-trisphosphate (IP3) mass (Estrada et al 2000).…”

“…These effects were characterized by an early increase in intracellular calcium, triggered 15-30 s after testosterone addition, preceded by an increase in inositol 1,4,5-trisphosphate (IP3) mass (Estrada et al 2000). In addition to the effects on IP3, exposure of myotubes to androgens produced an early, transient, IP3-calcium-dependent increase in extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation (Estrada et al 2003). The use of dominant negative mutants of Ras and MEK demonstrated that androgens activate the Ras/MEK/ERK pathway (Estrada et al 2003).…”

“…In addition to the effects on IP3, exposure of myotubes to androgens produced an early, transient, IP3-calcium-dependent increase in extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation (Estrada et al 2003). The use of dominant negative mutants of Ras and MEK demonstrated that androgens activate the Ras/MEK/ERK pathway (Estrada et al 2003). The actions described above were not blocked by cyproterone acetate, an inhibitor of the AR, indicating that distinct non-genomic pathways exist for the action of androgens in skeletal muscle cells (Estrada et al 2000(Estrada et al , 2003.…”