Since natural and recombinant glycoprotein hormones

Since natural and recombinant glycoprotein hormones exhibit some differences in their carbohydrate structures, we checked whether this could affect the permissive effect on FSK-induced cyclic AMP response in MLTC-1 cells. Fig. 6 shows that the same sensitizing effect to FSK is observed with pituitary hLH and natural or recombinant hCG but not with any of the non-human LHs or CG tested (rat, ovine, bovine, porcine, equine). Nevertheless, all non-human LHs and CG synergized with 10 µM FSK when present at the same time but not when preincubated before and eliminated at the time of FSK addition. Since sub-stimulating concentrations of human LH and CG sensitized MLTC-1 T7 High Yield Cy5 RNA to FSK, it was of interest to see how they affect subsequent stimulation of cyclic AMP accumulation by LHs and CGs themselves. Fig. 7 shows the concentration-response effect of a 20 min-preincubation with sub-stimulating concentrations of hLH (0–70 pM) on the subsequent response of MLTC-1 cells to a stimulating concentration of hLH (0.7 nM). The presence of non-stimulating concentrations of hLH during preincubation, led to a more precocious and higher cyclic AMP response to a stimulating concentration of hLH. Fig. 8 shows that the presence of non-stimulating concentration of hLH during a 20-min preincubation, led to a more precocious cyclic AMP response to FSK than when FSK and the non-stimulating concentration of hLH are added together. Nevertheless, FSK stimulation stopped after 15 min when the non-stimulating concentration of hLH had been removed before FSK introduction whereas it showed a much longer duration when FSK and hLH were present together. In order to point out a possible involvement of Gi protein in the synergy of non-stimulating concentration of LH with FSK, we studied the effect of preincubation of MLTC with pertussis toxin on the synergy of a sub-stimulating hLH concentration with 10 µM FSK. No difference was observed in the presence or absence of PTX (Fig. 9).
Discussion In the present report, we have compared the properties of Luteinizing Hormones (LH) and Chorionic Gonadotropin (CG) from various mammalian species in their ability to sensitize adenylate cyclase (AC) in MLTC cells, to forskolin (FSK) stimulation. Indeed, Leydig cells have been found to be unsensitive to 10 µM FSK alone but this concentration of the diterpene has been previously shown to synergize with low stimulating concentrations of hCG (Lefevre et al., 1985). Indeed, it has also been previously shown that FSK exhibits much lower affinity to ACs in the absence of Gs (40 µM vs 0.1 µM) (Dessauer et al., 1997). Human CG like LHs bind to the LH receptor (LHR). In numerous in vivo and in vitro experiments, hCG has been used as a substitute of LH because of its higher potency. In MLTC cells used in the present study, no significant stimulation of intracellular cyclic AMP accumulation was observed with 10 µM FSK (Fig. 1), and only a very marginal one with 100 µM FSK (not shown). The FSK lot used here, was found to strongly stimulate cyclic AMP accumulation at 10 µM concentration in Cov434 cells (Zhang et al., 2000) showing that it is fully active. We show here that sub-stimulating concentrations of various LHs or CGs (≤70 pM) together with a non-stimulating concentration of forskolin (10 µM FSK), are able together, to stimulate cyclic AMP accumulation in MLTC cells (Fig. 1, Fig. 2). Synergy of gonadotropins with FSK in adenylate cyclase stimulation has been described a long time ago (Lefevre et al., 1985, Mokhtari et al., 1988), but in the present work, the effect observed is more marked as neither the gonadotropins nor FSK, are stimulating alone at the concentrations used. Since both the gonadotropins and FSK are inactive alone at the concentrations used, it is clearly not an additive effect but, rather, a synergistic effect. Pertussis toxin did neither alter nor potentiate this synergy (Fig. 9), therefore it can be ruled out that Gi plays a role in it. This result can be matched with the previous observation that PTX had no effect on rat testicular Leydig cells from less than 15-day rats (Eskola and Huhtaniemi, 1996, Eskola et al., 1994). Unsurprisingly, this indicates that the MLTC which are tumor cells, are partly de-differentiated Leydig cells.