Release of thyroid stimulating hormone from chick anterior pituitary glands by thyrotropin releasing hormone (TRH)

Chicks two and ten days-of-age respond to a wide range of thyrotropin releasing hormone (TRH) dosages as measured by thyroid uptake of ³²P. The duration of hormone and ³²P action is important. Excellent responses were obtained with the injection of 1.0 μCi³²P at one hour and TRH either at one or four hours before autopsy in both two-day and ten-day-old birds. The ³²P uptake in the thyroid glands was increased by doses of hormone which ranged from 40 nanograms to 125,000 nanograms and was bimodal. Analysis of the data when calculated using log¹⁰ of dose was best accomplished by the use of 5th-degree polynomial equations. It is suggested that the bimodal response is a result of a dual action of TRH. First, TRH initiates the release of stored TSH from the anterior pituitary; and second, TRH stimulates the secretion of newly synthesized TSH by the anterior pituitary.     

Comparison between the biological effects of LH-RH and buserelin on the induction of LH release from hemi-pituitary glands of female rats in vitro

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.     

Comparison of mammalian luteinizing hormone releasing hormone (LH-RH), and of an analog (ICI 118630), on luteinizing hormone and ovarian steroid (progesterone, oestradiol) secretions in laying hens. (Gallus domesticus)

This experiment was conducted to compare the luteinizing hormone (LH), progesterone (P4) and oestradiol (E2) release in response to injections of various doses of synthetic mammalian luteinizing hormone-releasing hormone (LH-RH) and of an LH-RH agonist, ICI 118630, administered to laying hens 4 to 9 hours after a mid-sequence ovulation. Plasma LH increased significantly within 10 minutes of injection of either compound whereas any increases in plasma steroid concentrations were discerned later, at approximately minutes post-injection. No dose-response relationship was found for either compound with respect to LH release, but ICI 118630 appeared more potent than LH-RH. This analog also produced a greater mean incremental rise in plasma progesterone, but not oestradiol, than LH-RH, and this was found in animals injected at a time when the largest ovarian follicle was not mature. These result suggest that ICI 118630 is a more potent releasing hormone in the hen at the level of the pituitary, and that it may have a stimulating effect on ovarian progesterone secretion.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

Binding capacity of luteinizing hormone-releasing hormone and its analogues for pituitary receptor sites.

Competition for luteinizing hormone-releasing hormone (LH-RH) receptor sites by the inhibitory analog [D-Phe², D-Trp³, D-Phe⁶]-LH-RH and by the superactive stimulatory analog [D-Trp⁶]-LH-RH was observed in adenohypophysial homogenates incubated at 4°C. Competition for LH-RH binding sites was less evident with adenohypophysial plasma membranes. The binding affinities of these analogues to LH-RH pituitary receptors can explain at least in part their respective action in blocking ovulation and in inducing a greater release of luteinizing hormone and follicle stimulating hormone than the parent hormone.     

A comparison of the LH-releasing activities of LH-RH and its agonistic analogue buserelin in the ovariectomized rat

LH-RH and the potent agonistic analogue (D-Ser(But)6-des-Gly10)-LH-RH(1-9)-ethylamide (HOE-766 or buserelin) were at several doses either infused or injected intravenously in 5-weeks-ovariectomized rats, which had been treated with either 3 micrograms estradiol-benzoate (EB) or with oil, 24 h previously. Blood samples for assay of LH were taken during the subsequent 24 h. Pituitary glands were removed at the end of the experiments. Buserelin, when infused, was slightly more effective than LH-RH on releasing LH. When injected, however, buserelin was at the higher dose ranges increasingly more effective as an LH-releasing agent than LH-RH. EB-treatment increased the LH response of the pituitary gland to both peptides in an identical way. It was concluded that buserelin derives its high potency not from its intrinsic LH-releasing activity, which is only slightly greater than that of LH-RH, but from a longer duration of action.     

Resistance to enzymic degradation of LH-RH analogues possessing increased biological activity.

Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly⁶, and two additional analogues in which the Leu⁷ residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly⁶-Leu⁷ position, proved less effective in degrading all the analogues tested. Among the Gly⁶ substituted analogues, [D-Trp⁶] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.     

Synthesis and secretion of phosphorylated growth hormone by rat pituitary glands in vitro

Rat anterior pituitary glands were incubated in buffered medium, pH 7.4, containing 32Pi. After incubation the tissue and medium were separated and a post-mitochondrial supernate (PMS) of the tissue homogenate was prepared. Gel filtration of the PMS and medium resulted in a radioactive peak which coincided with the elution volume of authentic rat growth hormone (rGH). Polyacrylamide gel electrophoresis of the radioactive peak under denaturing condition resulted in a protein-staining band having the same mobility as authentic rGH. Autoradiography of the gels revealed radioactivity precisely at the position of growth hormone as well as elsewhere. The specific radioactivity of the PMS [32P]GH was estimated to be 5 to 10 times greater than that of tissue [32P]GH. These results indicate that phosphorylated GH is synthesized and secreted by pituitary glands in vitro.     

Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone

A peptide having gonadotropin-releasing activity was isolated in a yield of 2.5 μg from an extract of 2,000 chicken hypothalami. The biopotency was monitored using rat anterior pituitary cell culture system. The peptide differs from mammalian Luteinizing Hormone-Releasing Hormone (LH-RH) in its behavior during chromatographic separation (ionexchange and high performance liquid chromatography) and in its reaction towards anti-LH-RH antiserum directed against the C-terminal region of the LH-RH molecule. The peptide (chicken LH-RH) stimulates secretion of both LH and FSH from rat anterior pituitary cells. The biological potency of this peptide was about 4 % of that of the authentic decapeptide estimated in the rat anterior pituitary system. The amino acid composition is (Ser, Pro, Glx₂, Gly₂, Leu, Tyr, His, Trp), which differs from mammalian LH-RH only in that one Arg residue is replaced by a Glx residue. Based on the behavior on CM cellulose chromatography and the reaction towards anti-LH-RH antiserum, one possible structural candidate for this peptide (chicken LH-RH) is [Gln⁸]-LH-RH.     

Studies on the enzymic degradation of luteinizing hormone releasing hormone by rat pituitary plasma membranes

Degradation of luteinizing hormone releasing hormone (LH-RH) by purified plasma membranes from rat pituitaries was investigated. Synthetic LH-RH (0.5 mg/ml) was incubated (20 min, 37°C) with pituitary plasma membranes (750 μg protein/ml). The reaction was stopped by centrifugation at 4°C. The degradation products were isolated by high pressure liquid chromatography using a reversed-phase column. Amino acid analysis of the degradation products indicated that the N-terminal tripeptide (pGlu-His-Trp) and the N-terminal hexapeptide (pGlu-His-Trp-Ser-Tyr-Gly) sequence of LH-RH are the main degradation products. These results suggest that the main cleavage sites of LH-RH by the pituitary plasma membrane-bound enzymes are the Gly⁶-Leu⁷ and the Trp³-Ser⁴ bonds of the neurohormone.     

Inactivation of two hyperactive LH-RH analogues by rat hypothalamic peptidases.

Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are beleived to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself; the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TUB)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptidase with increased biological activity.     

Effects of LH-RH on isolated pituitary gonadotropic cells.

Rat anterior pituitary glands were dissociated with Pronase and the cells were separated by velocity sedimentation at unit gravity. After 30 min of incubation of the enriched gonadotropic cells with LH-RH, there was a significant increase in LH and FSH in the incubation medium. LH-RH (100 ng/ml) and 10(-3) M cAMP both caused significant increases in LH in the incubation medium after 24 hr of incubation.     

Effect of pre-incubation with different concentrations of LH-RH on subsequent LH release caused by supramaximally active amounts of LH-RH; role of LH-RH-induced protein synthesis.

Anterior pituitary glands from intact diestrous female rats were incubated for two consecutive periods of 3 hours. During the first period various submaximally active amounts of luteinizing hormone-releasing hormone (LH-RH) were added to the media, whereas during the second period a supramaximally active concentration of LH-RH was present. When during the second incubation period protein synthesis was inhibited by cycloheximide, the amount of luteinizing hormone (LH) released during that period was positively correlated to the concentration of LH present during the first incubation period. This relationship was not seen when cycloheximide was absent, or when cycloheximide was present throughout both periods. Total LH was not affected by LH-RH; thus no effect of LH-RH on LH synthesis was observed. It is concluded that the amounts of protein synthesized by the pituitary glands in response to the different amounts of LH-RH during the first incubation period can constitute a limiting factor for the response to the supramaximally active amount of LH-RH added during the second incubation period.     

Progesterone and LH variations after LH-RH administration in the luteal phase of the menstrual cycle

We have investigated the pituitary and luteal responses to LH-RH and their related changes. 11 normal women were studied during the luteal phase (day +4/+11). Blood samples were collected every 15 min for a basal period of 180 and 120 min after the intravenous administration of 25 micrograms of LH-RH. Progesterone (P) and LH were assayed by radioimmunoassay. Data were analyzed as maximum peak and its percent increase (delta max), integrated secretory area (ISA) and percent increase of ISA (delta A) in respect to basal values for both P and LH. LH-RH elicited a secretory response of both hormones in all cases. ISA of LH was significantly greater after LH-RH administration in respect to basal values (p less than 0.001) and delta max accounted to 475 +/- (SE) 36% of the basal concentration. Luteal responsiveness varied from about 115-130% to more marked increments. ISA of P differed from basal to stimulated conditions (p less than 0.05) and delta max was 166 +/- (SE) 14%. The analysis of temporal relationship between P and LH secretion showed that LH promptly rose after LH-RH, while the enhancement of P plasma levels occurred within 31 +/- 19 min after LH rise. Then P levels reached a plateau, values of which were statistically different from those observed before LH-RH administration. In two cases where luteal function was blunted or absent, in spite of marked increments of LH, P secretion did not occur. These data are consistent with the presence of close relationships between hypothalamic, pituitary and luteal functions and strengthen the contention about the usefulness of LH-RH during luteal phase for the lifespan and maintenance of corpus luteum.     

The role of protein kinase C in LH release

To investigate the postreceptor mechanism, especially the role of protein kinase C (C-kinase), in luteinizing hormone (LH) release from anterior pituitary cells, dispersed rat anterior pituitary cells were stimulated with luteinizing hormone-releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin), 12-0-tetradecanoyl phorbol-13-acetate (TPA) and trifluoperazine (TFP) and the LH released into the medium was determined by radioimmunoassay. LH released by combined stimulation with TPA and either LH-RH or Buserelin was significantly less than that released by LH-RH or Buserelin alone (LH-RH: p less than 0.05; Buserelin: p less than 0.01). It is thought that this paradoxical phenomenon occurred due to desensitization accompanied by down-regulation of LH-RH receptors induced by TPA. This hypothesis was supported by the finding indicating that the binding capacity of LH-RH receptors decreased in a time-course manner during incubation with TPA. The amount of LH released by combined stimulation with TPA and TFP was significantly greater than with TPA alone (P less than 0.01). This suggests that TFP has dual actions, i.e., facilitating and inhibiting LH release.     

A study of TSH-synthesis of spontaneously hypertensive rats by electron microscopic morphometry and autoradiography

Summary In order to compare the functional state of the anterior pituitary of spontaneously hypertensive rats (SHR) with that of normotensive Wistar Kyoto rats (WKR), the anterior pituitary was examined by morphometry and autoradiography at the level of electron microscopy. The relative number and the relative volume of thyrotrophs in the anterior pituitary were significantly greater in SHR compared with age-matched WKR at 0, 7, 30–33 days, and 10 months of age, while the relative number of somatotrophs in SHR was significantly smaller at 1 and 10 months of age. Electron microscope autoradiographic analysis of uptake of ³H-lysine by thyrotrophs of both strains at the age of approximately one month showed that ³H-lysine was incorporated into protein and transported finally to secretory granules which migrated to near the cell membrane to be discharged. Silver grains were significantly more numerous over the thyrotrophs of SHR than over those of WKR at 30 min, 1 h, and 4h after the injection of ³H-lysine.The present study has ascertained morphologically that a congenital hypersynthesis of TSH by the anterior pituitary occurs in SHR.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Feedback control of FSH secretion in the male rat

Site of feedback control of FSH secretion in the male rat was studied by measuring changes in serum LH, FSH and hypothalamic LH-RH by radioimmunoassay in rats after castration and after 500 rad X-irradiation to the testis. The rise in serum LH and FSH in castrated animals was associated with a significant fall in hypothalamic LH-RH 16 and 24 days after castration. Serum FSH rose significantly after X-irradiation without a significant change in serum LH or hypothalamic LH-RH content up to 30 days after irradiation. When pituitary halves from X-irradiated animals were incubated in vitro in the presence or absence of synthetic LH-RH, there was a significant rise in FSH (but not LH) released in the incubation medium in the absence of added LH-RH. The response of the pituitaries to LH-RH was, however, not different between control and irradiated rats. It is concluded that the testicular FSH-inhibitory substance acts predominantly at the pituitary gland on the LH-RH independent release of FSH.     

Self-priming effect of LH-RH on pituitary gonadotropins in hyperprolactinemic women

To investigate how various concentrations of serum prolactin (PRL) influence the priming effect of luteinizing hormone releasing hormone (LH-RH) on the pituitary gland, 24 women with various blood PRL concentrations received intravenous injections of 100 micrograms of synthetic LH-RH twice at an interval of 60 minutes and their serum LH and follicle-stimulating hormone (FSH) were measured and analysed. In the follicular phase with a normal PRL concentration (PRL less than 20 ng/ml, n = 6), marked first peaks of the two hormones following the first LH-RH stimulation and enhanced second peaks after the second LH-RH administration were observed, indicating a typical priming effect of LH-RH on gonadotropins, though the second response of FSH was more moderate than that of LH. In hyperprolactinemia, in which the serum PRL concentration was higher than 70 ng/ml (n = 13), the basal concentration of gonadotropins was not significantly changed but the priming effect of LH-RH on LH and FSH was significantly decreased (p less than 0.01). No marked second peaks of LH and FSH were observed, suggesting an inhibitory effect of hyperprolactinemia on the second release of LH and FSH. In contrast, this effect was restored in a group of women whose serum PRL concentration was between 30 and 50 ng/ml (n = 5). Furthermore, enhanced second peaks of both LH and FSH were noted after successful bromocriptine therapy reduced hyperprolactinemia (PRL greater than 70 ng/ml) to less than 25 ng/ml (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypophysial portal vascular infusion of TRH in the rat: an ultrasturctural and radioimmunoassay study.

The hypophysial portal vessels and anterior pituitary gland of adult male Wistar rats were exposed surgically. A hypophysial portal vessel was cannulated and infused for one minute with saline or thyrotrophin (TRH). Anterior pituitary glands were collected at 1,5,15,30 or 60 minutes after cessation of infusion, for light and electron microscopic examination. Before and immediately after cannulation of a portal vessel, a 1-ml sample of blood was collected at 1,5,15,30, or 60 minutes, from the femoral vein for radioimmunoassay (RIA) of growth hormone. Thyrotrophs from anterior pituitary glands of rats infused with TRH displayed emiocytic activity at all time-periods studied. Rough endoplasmic reticular (RER) cisternae were dilated at 15 minutes following infusion and remained dilated at 30 and 60 minutes. TRH was observed to stimulate emiocytic activity in most pituitary cell-types. Extensive dilations of RER cisternae were also observed in mammotrophs and gonadotrophs, but were not observed in somatotrophs or adrenocorticotrophs. The demonstration that thyrotrophs, mammotrophs, somatotrophs, and gonadotrophs respond to TRH suggests that some common features may be shared by these cells. Preliminary analysis of the RIA data show that TRH was potent in elevating radioimmunoassayable growth hormone levels. Significant increases (p less than 0.02) in plasma GH levels were present at the earlier time periods studied (1,5, and 15 minutes) following the infusion of TRH, but no at 30 or 60 minutes. These findings provide additional support for the non-specific action of TRH upon hte various adenohypophysial cell types, and demonstrate that TRH stimulates these cells by a direct action on the adenohypophysis.