Rat intestinal cells prepared from testes were incubated in the presence of different lutropin derivatives obtained by chemical modification of the amino groups. The cAMP accumulation and the testosterone biosynthesis were determined in the cell homogenates. Binding determinations were carried out by a radioligand receptor assay using tritiated methylated lutropin. The binding activities \ p=m-\ relative to native LH \ p=m-\ of three different derivatives obtained by reductive alkylation (methylated, ethylated and isopropylated LH) were in good agreement with the relative potencies assessed by their capacity to stimulate cAMP and testosterone production. Guanidinated LH (11 \p=m-\ NH2 groups modified) exhibited a binding activity and a relative potency relatively high with regard to cAMP accumulation (as compared with that of native LH). Its steroidogenic potency, however, was very low. When Leydig cells were incubated in the presence of native and guanidinated LH, the testosterone production was similar to that induced by the derivative alone, indicating that the derivative exerted a competitive inhibitory action preventing the stimulation of steroidogenesis by native LH. These results suggest that a guanidinated derivative is able to bind to the LH receptor and the complex so formed is able to be coupled with an adenylate cyclase pool (or cAMP compartment) which is not connected with the steroidogenic pathway.Chemical modification of the amino groups of lysine residues can be achieved in lutropin (luteiniz¬ ing hormone, LH) without loss or with only a partial loss of the biological activity, if the positive charge of these residues is not abolished (De la Llosa et al. 1974a; Liu et al. 1974). These derivatives have been tested by various bioassays, in vivo and in vitro. It has been shown that they are able to bind to the LH-receptors (Liu et al. 1974; De la Llosa-Hermier et al. 1977), to activate ovarian adenylate cyclase (Tertrin-Clary & De la Llosa 1978) and to stimulate steroidogenesis in the ovary. We considered it of interest to investigate the correlation between the succes¬ sive responses induced by these derivatives which are known to be consecutive steps in the mecha¬ nism of action of LH. Rat Leydig cells were used as biological material. As is well known, binding of lutropin to the receptors of Leydig cells is followed by activation of adenylate cyclase, and the subse¬ quent cyclic AMP accumulation is accompanied by stimulation of testosterone production. The data reported here show that a good correlation is observed for most of these derivatives in each of the three steps, but that at least in one case (guanidinatedLH), binding to the receptors and stimula¬ tion of adenylate cyclase do not result in a propor¬ tional increase in testosterone biosynthesis. In such a case an inhibitory effect of the derivative on the final action of the hormone could be expected and was demonstrated. Material and MethodsOvine LH was prepared in a highly purified state (biolo¬ gical potency: 2 x LH-NIHS11) in our labo...
The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon.
Biological activities of several derivatives of ovine LH obtained by chemical modification of the amino groups were investigated using ovaries from pseudopregnant rats. Binding-inhibition activities and steroidogenic potencies of ethylated, isopropylated and guanidinated LH were in good agreement, whereas adenylate cyclase activities were relatively greater. When compared with previous results on binding-inhibition activities and steroidogenic potencies using isolated rat Leydig cells, the ovaries from pseudopregnant rats appeared to be more discriminating. Ethylated and isopropylated derivatives exhibited lower binding-inhibition activities and steroidogenic potencies in female gonads. This difference was particularly evident in the case of guanidinated LH which exhibited a very low binding-inhibition activity and consequently was unable to act as an inhibitor of the action of LH on the ovaries. Guanidinated porcine LH (in which all the lysine residues of the alpha-subunit were transformed into homoarginine, without modification of the beta-subunit which does not contain lysine) showed similar biological activities to guanidinated ovine LH in the isolated Leydig cells as well as in pseudopregnant ovaries. It can, consequently, act as an inhibitor of LH action on Leydig cells but not on the ovary of the pseudopregnant rat. Thus, the inhibitory properties of this derivative can be ascribed to the modification introduced in the alpha-subunit.
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