Modifications of Luteinizing Hormone Biosynthesis and Release by Gonadotropin-Releasing Hormone, Cycloheximide, and Actinomycin D*

Abstract

In the first experiment, the influence of synthetic gonadotropin-releasing hormone (GnRH) on the time course of [3H]glucosamine ([3H]GA) and [14C]alanine ([14C]A) incorporation into LH by quartered rat anterior pituitary glands and the sequence of release of radiolabeled and total immunoreactive LH (IR-LH) was investigated. Radiolabeled LH was measured by immunoprecipitation and total IR-LH was measured by RIA. After lag periods of 5 and 20 min, respectively, incorporation of [14C]A and [3H]GA into tissue LH increased linearly for 4 h. GnRH stimulated incorporation of [3H]GA only. The ratio of [14C]A-LH to [3H]GA-LH (14C:3H ratio) in the tissue decreased significantly with time and with GnRH treatment. In the second experiment, replenishment of GnRH in the medium every 0.5 h elevated the release rate (release during each sequential 0.5 h) of both [3H]GA and [14C]A-labeled LH within 1.5 h. The release rate of radiolabeled LH increased linearly until 3.5 h. The 14C:3H ratio in LH released during each time interval was reduced by GnRH. The release rate of IR-LH increased linearly with time, plateaued by 1.5-3 h, and started to decline. In other experiments, cycloheximide blocked synthesis of [14C]A-LH and greatly reduced the GnRH-induced synthesis and release of [3H]GA-LH, but reduced release of IR-LH by only 25%. Actinomycin D had no effect on GnRH-induced synthesis and release of LH at 2 h, but significantly reduced both at 4 h. These data suggest that 1) the time course for the release of preexisting IR-LH differs from that for newly synthesized LH, 2) the newly synthesized LH released in response to high levels of GnRH has more sugar residues than that released under basal conditions, 3) the GnRH-induced LH release can occur under conditions in which LH synthesis has been blocked, and 4) synthesis of messenger RNA is not required for GnRH-induced LH release or short term LH synthesis but seems to be required for continued synthesis and subsequent release of LH.

Phorbol Ester-Induced LH Release in Pituitary Gonadotrophs: Effects of Antagonists of Calmodulin and GnRH.

“…One possible explanation of this unexpected synergism between calmodulin antagonists and TPA is that cytosolic Ca2+ is made more available for activation of protein kinase C during the inhibition of calmodulin by the antagonist [31][32][33]. It is possible that the inhibition of PDE by calmodulin antagonists increases the cyclic nucleotides which have been implicated in the stimulation of gonadotropin synthesis [9,34]. However, neither cyclic AMP nor cyclic GMP appears to participate in LH release within a couple of hours [35].…”

Biphasic release of thyrotropin in response to thyrotropin-releasing hormon (TRH) from rat anterior pituitary cells in vitro: Possible dependence on protein synthesis.

“…This is the first demonstration that the phase II release of TSH involves protein synthesis. Though the protein synthesisdependent release in LH has been reported (Pickering and Fink, 1976 ;Kercret and Duval, 1978 ;Liu and Jackson, 1978 ;Bourne and Baldwin, 1980), the significance of the dependency in the dynamics of the hormone secretion is still unknown. Since the shift from phase I to phase II release occurs before complete depletion of a previously prepared-releasable pool, it is possible that the phase II release in an early period is also consuming the releasable pool that had been prepared.…”

“…Since the profile of the biphasic release caused by a maximally effective concentration of TRH was different from that by high K+ (60 mM) solution, there seems to be a specific action of TRH related to the biphasic release (Shiota et al, 1984). Though protein synthesis is theoretically expected to play an important role in the second-phase release, as shown in the case of LH (Pickering and Fink, 1976 ;Kercret and Duval, 1978 ;Liu and Jackson, 1978 ;Bourne and Baldwin, 1980), the relation of the first and the second release of TSH in connection with protein synthesis is poorly understood.…”

Actinomycin D Affects Thyrotropin-Releasing Hormone-Induced Heterogeneous Forms of Glycosylated Thyroid-Stimulating Hormone in Rat Anterior Pituitary

“…in the presence of 10 ng/m l TRH (Tanabe Pharmaceutical Co., Osaka, Japan), cycloheximide (25 jig/ml), or actinomydn D (25 ug/ml). Although in the previous studies varying doses (ranging from 0.1 to 100 pg/m l) of cycloheximide and actinomydn D were used to inhibit pituitary protein synthesis [16,17,23], we employed 25 pg/m l of each drug, a dose suita ble for significant inhibition o f protein synthesis [25,26]. In the present study, cycloheximide and actinomydn D at doses o f 25 pg/m l were observed to significantly inhibit [l4C)alanine incorporated into pituitary protein which was precipitated by trichloroacetic acid (3 h incubation, the TRH-added group, 22.7 ± 1.7; the TRH + cycloheximide-added group, 2.1 ± 0.4; the TRH + actinomydn D-added group, 13.3 ± 1.1; 6 h incubation, the TRH-added group, 31.4 ± 3.7; the TRH + cycloheximide-added group, 1.8 ± 0.1; the TRH + actinomycin Dadded group, 19.1 ± 1.4 x 10-3 dpm /A P, n = 5 in each group).…”

“…This drug was also found to be unable to change gonodotropin-releasing hormone (GnRH)-stimulated LH release [23], It was observed that actinomycin D did not inhibit [3H]glucosamine-incorporation into pituitary hormones in the presence ofTRH and GnRH. while cycloheximide, a drug inhib iting protein synthesis [24], significantly reduced its incorpora tion [25,26]. These observations were interpreted to support the possibility that messenger RNA may not contribute to the car bohydrate synthesis of glycoprotein hormone in the short-termincubation, although protein synthesis is required for glycosyla tion of pituitary hormone.…”