We have investigated the effect of 5'-guanylylimidodiphosphate (Gpp(NH)p) and two disulfide bond reducing agents, reduced glutathione (GSH) and dithiothreitol (DTT), on the modulation of [I-125-Tyr(10)]hGRF(1-44)NH2 binding to GRF receptor binding sites, in pituitaries of young and aging rats. In pituitaries from 2-month-old rats, Gpp(NH)p (0.1-1.0 mM), GSH and DTT (1-50 mM) exhibited a partial but concentration-dependent inhibitory effect on GRF specific binding. These effects were associated with a conversion of the high affinity GRF binding sites to lower affinity sites and to a reduction of the apparent number of total binding sites (high and low). No potentiation of these effects was observed when Gpp(NH)p (1 mM) and DTT (1 mM) were combined. In pituitaries from 14-month-old rats, Gpp(NH)p (1 mM) was capable of modulating GRF binding parameters in a similar fashion to that in pituitaries from 2-month-old rats. In pituitaries from 18-month-old rats, the high affinity GRF binding sites were already blunted and neither Gpp(NH)p nor Gpp(NH)p plus DTT significantly altered GRF binding parameters. In addition, in 20-month-old rats, the affinity of hCRF(1-29)NH2 and that of the full antagonist N-alpha-Ac-[D-Arg(2),Ala(15)]rGRF(1-29)NH2 were respectively decreased 9.3- and 9.9-fold. Our results suggest that in aging, alterations of GRF receptor binding sites could involve disulfide bond reduction or other structural modifications leading to conformational changes, similar to those induced by GSH or DTT. Such structural changes may prevent an efficient coupling of the GRF receptor with its ligands and G-protein, leading to a loss of somatotroph responsiveness.
