Basal and gonadotrophin-releasing hormone-induced biosynthesis and release of luteinizing hormone: Effect of calcium deprivation

Abstract

The present study examines the basal and gonadotrophin-releasing hormone (GnRH)-stimulated biosynthesis and release of luteinizing hormone (LH) by pituitary cells in primary culture, and the effect of extracellular calcium deprivation on these events. Pituitaries from ovariectomized adult rats were enzymatically dispersed and cultured for 96 h. The cells were then incubated for 5 h (Expts. 1 and 3) or for different time intervals between 0 and 5 h (Expt. 2), in medium containing [ 14C]leucine ([ 14C]leu) and [ 3H]glucosamine ([ 3H]gln), with or without GnRH. Total immunoreactive LH (iLH) was measured in the medium and the cell extract by radioimmunoassay. LH translation (as estimated by [ 14C]leu incorporation into LH; [ 14C]LH) and LH glycosylation (as estimated by [ 3H]gln incorporation into LH; [ 3H]LH) were measured by immunoprecipitation with specific LHβ antiserum in both medium and cell extract. Treating the cells with GnRH caused both time- and dose-dependent increases of iLH in the medium as well as in total (cells plus medium) content, with an approximate ED 50 of 0.7 nM. GnRH also stimulated LH biosynthesis by increasing both LH polypeptide chain synthesis and LH glycosylation. The effect of GnRH on LH glycosylation was detected earlier than that on translation, the [ 3H]LH rates of production and release being higher than those of [ 14C]LH. These findings suggest that GnRH-induced translation and glycosylation of LH are independently regulated. Removal of extracellular calcium resulted in the loss of cellular responsiveness to GnRH, preventing not only the stimulatory effects of GnRH on total and released iLH but also the GnRH-induced incorporation of both [ 14C]leu and [ 3H]gln into newly synthesized LH. These observations suggest that GnRH-stimulated LH glycosylation and LH translation involve calcium-dependent mechanisms. Neither the uptake of radiolabeled precursors nor their incorporation into total protein were affected by GnRH or Ca 2+-deficient (no added calcium) medium. The results also suggest that the release of newly synthesized LH is regulated differently from previously synthesized stored hormone.