A transplantable rat leydig cell tumor—3. Isoproterenol and prostaglandin E 1 (PGE 1) responsive adenylyl cyclase (AC). regulatory effects of nucleotides and magnesium

Abstract

The activity of the membrane bound adenylyl cyclase (AC), the effects of nucleotides, Mg 2+-cations and its responsiveness to isoproterenol and prostaglandin E 1 (PGE 1) were examined in a transplantable rat Leydig cell tumor (H-540). Both isoproterenol and PGE 1 caused activation of the AC in Leydig cell tumors. The degree of activation by PGE 1 (4–5-fold) was approximately twice that of isoproterenol (2–3-fold). The addition of both AC agonists simultaneously was not additive indicating that they activate AC of the same cell. Increasing concentrations of ATP (0.025–2.0 × 10 mM) caused a concentration dependent increase in both the basal and hormone stimulated AC activity, and the activation by isoproterenol and PGE 1 (relative response) revealed a slight but significant increase with increasing ATP concentrations. Lineweaver-Burke analysis of these data indicated an apparent K m for ATP (Mg ATP) of 0.16 mM. Free magnesium did not influence the apparent K m of the AC for ATP. Increasing concentrations of free Mg 2+ (0.24–13.2 mM) also caused a concentration dependent increasing activation of AC activity up to a concentration of approx 6 mM in excess of Mg 2+-binding ingredients. Higher concentrations of free Mg 2+ (13.1 mM) caused a small but significant decrease in both basal and agonist stimulated AC activity. In contrast to other reports, activation by isoproterenol and PGE 1 was in general not influenced by the concentration of Mg 2+. Both GTP and GMP-P (NH)P stimulated basal and hormone stimulated AC activity ( K act 1 μM), but with different kinetics. In the presence of GTP, AC activity was almost constant for 90 min. In the presence of GMP-P(NH)P, AC activity was much higher, but constant AC activity occurred after a certain lag time (7–10 min), which was reduced by PGE 1 and isoproterenol. In conclusion, cAMP production in Leydig cell tumors is stimulated by both PGE 1 and isoproterenol. The AC activity and activation by these agonists are regulated by Mg 2+ and nucleotides in a slightly different manner from most other cells. The association between AC activation and stimulation of steroid production by Leydig cell tumors remains to be investigated.