Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells.

Abstract

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.