A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KC1 resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.     

Studies on the properties of hypothalamic luteinizing hormone-releasing hormone

The effects of various chemical and enzymatic treatments on the biological activity of porcine luteinizing hormone-releasing hormone (LH-RH) are described. This experiment was performed before the elucidation of the structure of LH-RH. LH-RH activity was abolished by the following endopeptidases: chymotrypsin, subtilisin, papain, and thermolysin, but not by pepsin or trypsin. Exopeptidases did not affect LH-RH activity, but a purified preparation of pyrolidone carbosylpeptidase did. The amino acid sequence of LH-RH/FSH-RH was established to be (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Amine. This decapeptide lacks both the Amine terminus and the COOH terminus. Its Amine-terminal dipeptide sequence,(pyro)Glu-His, is similar to that of tyrotropin-releasing hormone. The lack of inactivation by the exopeptidases is in good agreement with these findings. Treatment with various chemical reagents showed that tyrosine, histidine, tryptophan, and arginine in LH-RH are important for its biological activity. Nitrous acid and Edman degradation did not inactivate LH-RH. These results are also in agreement with the determined structure of LH-RH. This hormone showed a high follicle-stimulating hormone-releasing hormone (FSH-RH) activity. The inactivation of LH-RH was always accompanied by a loss of FSH-RH activity. These experiments also shed some light on the structure-activity relationship of this hormone.     

Regulation of the hypothalamic--pituitary--ovarian axis in women.

To account for the regulation of cyclic gonadotrophin release, the separate and interactive effects of the hormonal variable at the levels of CNS-hypothalamus, the pituitary and the ovary have been reviewed. The pituitary gonadotrophs, as target cells exhibited a remarkable cyclic change in their capacity which was correlated with the oestradiol levels. The ultimate release is determined by the relative size of the two pools' releasable gonadotrophins which are themselves regulated by the relative inputs of LH-RH and oestradiol, respectively. LH-RH appears to serve as a primary influence on the gonadotroph, stimulating gonadotrophin synthesis, storage and release. Oestradiol, for the most part, amplifies the action of LH-RH and induces the development of a self-priming effect of LH-RH, except that it impedes LH-RH-mediated gonadotrophin release. The pituitary capacity increases several-fold from the early to late follicular phase, and this is considered to be the prerequisite for the development of a mid-cycle surge. CNS-hypothalamic dopamine, norepinephrine, prostaglandins as well as LH-RH systems are involved in the negative and positive feedback effect of oestradiol. The possible steps and interactive elements in the triggering of LH-RH release for the initiation of the mid-cycle LH/FSH surge are considered.     

Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis

The development of the luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists and the principles of their clinical use were reviewed. In the 28 years that have elapsed since the elucidation of the structure of LH-RH, various applications in gynecology, reproductive medicine, and oncology have been established for LH-RH agonists and antagonists. These clinical applications are based on inhibition of the pituitary and the gonads. The advantage of the LH-RH antagonists is due to the fact that they inhibit the secretion of gonadotropins and sex steroids immediately after the first injection and thus achieve rapid therapeutic effects in contrast to the agonists, which require repeated administration. LH-RH antagonists should find applications in the treatment of benign gynecologic disorders and benign prostatic hypertrophy and in assisted reproduction programs. The primary treatment of advanced androgen-dependent prostate cancer is presently based on the use of depot preparations of LH-RH agonists, but antagonists like Cetrorelix already have been tried successfully. Antagonists of LH-RH might be more efficacious than agonists in treatment of patients with breast cancer as well as ovarian and endometrial cancer. Recently, practical cytotoxic analogs of LH-RH that can be targeted to LH-RH receptors on tumors have been synthesized and successfully tested in experimental cancer models. Targeted cytotoxic LH-RH analogs show a great promise for therapy of prostate, breast, and ovarian cancers.     

Substrate specificity of peptidases catalyzing LH-RH degradation in hypothalamus, liver and heart of rat]

Degradation of [125I]-LH-RH by peptidases of hypothalamus, liver and heart of the rat and the effects of LH-RH, LH-RH antagonist D-Phe2-Phe3-D-Phe6-LH-RH, LH-RH5-10, oxytocin, bradikinin, thyrotrophin-releasing hormone, melanocyte-inhibiting factor, luteinizing, follicle-stimulating, lactogenic and growth hormones were studied. It was shown that the degradation was inhibited with maximal efficiency by non-labelled LH-RH (Ki = 1,7--2,0 . 10(-6) M). This observation is indicative of peptidase specificity for LH-RH. It is assumed that specific peptidases of liver and heart are involved in the reglation of LH-RH concentration in these organs.     

Augmentation, by naloxone, of the frequency and amplitude of LH-RH pulses in hypothalamo-hypophyseal portal blood in the castrated ram

The effect of naloxone administration on the LH-RH secretion in hypophyseal portal blood and LH secretion in peripheral blood was studied in four short term castrated rams (between 2 to 4 days after castration). For two animals (A and B) given a single naloxone injection, an increase of LH-RH pulse amplitude was observed (A, 22.3 to 80.5 pg/ml and B, 22.5 to 34.5 pg/ml) with only a small (nonsignificant) increase in LH-RH pulse frequency. For animals C and D given four injections of naloxone, both LH-RH pulse amplitudes and LH-RH pulse frequency were increased. Means of LH-RH pulse amplitude increase from 29.3 to 65.1 pg/ml and from 34.6 to 50.8 pg/ml for animals C and D respectively and the number of LH-RH pulses detected during the 3 hrs. before and after the first injection of naloxone were respectively 3 vs. 5 and 3 vs. 7. Whereas all LH pulses were preceded with a LH-RH pulse in animals A and B, after the multiple naloxone injections in animals C and D, a rapid LH-RH pulse frequency was associated with a sustained increment of LH secretion in peripheral blood in such a way that individual LH pulses were not clearly defined. The present report is the first documentation on naloxone increasing the release of LH-RH secretion in hypophyseal portal blood of conscious, unrestrained, short-term castrated rams. The results indicate: (1) that the opiate antagonist naloxone is able to increase both the amplitude and the frequency of LH-RH discharge by the hypothalamus and (2), when the LH-RH pulse frequency exceeds one pulse every 30 min., discrete LH secretory episodes are not observed in peripheral blood.     

Antitumor effects of analogs of hypothalamic hormones in endocrine-dependent cancers

A new approach to the treatment of endocrine-dependent tumors based on analogs of hypothalamic hormones is in the early stages of development, but appears promising and significant. Administration of hypothalamic hormones can mimic hypophysectomy and gonadectomy, and is essentially devoid of side effects. A successful use of agonistic analogs of LH-RH for treatment of endocrine-dependent prostate cancer has been documented in several hundred patients. Experimental studies suggest that agonists and/or antagonists of LH-RH might be useful for treatment of breast cancer and pituitary tumors. Our work in animal models also indicates that analogs of somatostatin, alone or combined with LH-RH agonists, could be considered for therapy of chondrosarcomas, osteosarcomas, and pancreatic cancer. Experiments are in progress on the use of LH-RH analogs for treatment of ovarian cancer, neoplasms of the female genital tract, and for protection against gonadal damage during chemotherapy. These investigations should extend the concepts of endocrine treatment of cancers.     

Inhibitory control of the pituitary LH secretion by LH-RH in male rats.

Luteinizing hormone-releasing hormone (LH-RH) was administered to prepubertal male rats (intact, castrate or castrate-adrenalectomized, 60 g body weight) for 28 days (1 microgram LH-RH/day, s.c.), at a 10-fold physiological dose, as compared to the minimal FSH-releasing dose of 100 ng/rat s.c. In intact rats, serum LH and weight of androgen-dependent organs (vented prostate, seminal vesicles) were reduced after 14 days of treatment. In castrate rats, the postcastration rise in serum LH was abolished by treatment. Pituitary LH content, FSH secretion and prolactin secretion were not suppressed. Hypothalamic LH-RH was increased at 14 and 21 days. In castrate adrenalectomized male rats, LH secretion was also suppressed by 1 microgram LH-RH s.c. x 28 days. The hypothalamic LH-RH content did not increase. The pituitary LH-RH receptor level was not down-regulated after 14 days treatment either in intact or castrate male rats. Pituitary inhibition (LH release) in rats by a supraphysiological dose of LH-RH given for 28 days indicates that the optimal regime for chronic treatment has to be determined by monitoring LH release at regular intervals. Direct pituitary inhibition by LH-RH may explain some of the unexpected antifertility effects observed with high doses of LH-RH.     

The effects of luteinizing hormone releasing hormone (LH-RH) in pregnant rats. I. Postnidatory effects.

The effects of LH-RH on pregnancy in rats were investigated. A single 500 mcg injection of LH-RH on Days 9, 10, or 11 of pregnancy terminated pregnancy, whereas injection on Days 6-8 or 13-16 had little or no effect. The ED 50 on Day 10 for b.i.d. administration was 150 mcg and 550 mcg for a single injection. Administration on Day 9 was followed by a decrease in circulating progesterone levels on Days 10 and 11. The administration of large doses of progesterone reversed the effects of LH-RH administration on Days 7-12. Treatment with estradiol-17beta did not potentiate the effect of progesterone, but appeared to slightly retard fetal resorption when administered alone. The results suggest that the antifertility effect of LH-RH is mediated via functional luteolysis.     

Ethanol inhibits the naloxone-induced release of luteinizing hormone-releasing hormone from the hypothalamus of the male rat

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.     

Gonadotrophin-releasing hormone treatment for induction of follicular maturation and ovulation in amenorrhoeic women with anorexia nervosa.

Follicular maturation and ovulation can be induced in amenorrhoeic women with anorexia nervosa by long-term treatment with 500 mug of luteinizing hormone releasing hormone (LH-RH) every eight hours. In some women, however, treatment with LH-RH alone results in ovulatory menstrual cycles with indications of luteal phase insufficiency. Human chorionic gonadotrophin (HCG) was therefore given with LH-RH during three treatment cycles. This resulted in ovulation and normal corpus-luteum function, as shown by the occurrence of a single pregnancy in the only involuntarily sterile patient. During the prolonged LH-RH treatment the LH response to LH-RH increased in parallel with the increased oestrogen secretion while the follicle-stimulating hormone response to LH-RH decreased. These changes in the pituitary responsiveness to LH-RH may result from modulating effects on the pituitary by the sex steroids.     

The LH-RH system of the male European starling: photoperiod induces changes to a possible multifunctional peptide system

In many birds reproduction is triggered by long daylengths but, paradoxically, continued exposure to long days leads to photorefractoriness and a complete shut down of the reproductive system. As these effects are thought to be mediated through the secretion of LH-RH, immunocytochemical techniques were used to investigate changes in the LH-RH system when European starlings were exposed to different photoperiods. Starlings exposed to 11L:13D and with mature testes show strong immunostaining both of LH-RH perikarya and fibers. Photosensitive short-day (8L:16D) starlings with undeveloped testes show an almost identical distribution of strongly immunoreactive perikarya but with less dense fibre staining. However, long-day (18L:6D) photorefractory starlings with fully regressed testes, show a profound reduction in LH-RH immunostaining. Perikarya have the same distribution but show a much reduced intensity of staining and fibers had almost entirely disappeared from all regions of the brain. Preliminary observations on the ultrastructure of immunocytochemically identified LH-RH neurones are also reported.     

Acute stimulatory effects of an LH-RH agonist on the ovary in pseudopregnant rats

Pseudopregnant rats were anaesthetized on day 5 of pseudopregnancy with pentobarbital and hypophysectomy or sham operation was performed. Polyethylene cannulas were inserted into one of the femoral arteries and utero-ovarian veins. Five-minute blood samples were collected from the ovary for 40 minutes. Following the first five minute blood fraction LH-RH, an agonistic analogue (D-Phe6, des Gly-NH10(2)-LH-RH-ethylamide) or an antagonistic analogue (Formyl-D-Tryp1,3, D-pCl-Ph2, D-Phe6LH-RH) were superfused into the ovarian bursa for 5 minutes in a volume of 50 microliters. Blood pressure, ovarian venous outflow and haematocrit were continuously measured. From the blood samples progesterone and oestradiol-17 beta were determined by RIA and the secretion rate was calculated. It was found, that LH-RH and its antagonistic analogue has no effect on the blood flow, progesterone (P) and oestradiol-17 beta (E2) secretion of ovary. However, an agonistic analogue of LH-RH induced a rapid elevation of blood flow, diminished vascular resistance in the ovary, and in a dose of 50 ng increased the secretion rate of E2. In sham operated animals the effects of agonistic analogue was similar to the effects of 1.0 IU of hCG.     

A radioimmunoassay specific for [Gln]LH-RH: Application in the confirmation of the structure of chicken hypothalamic luteinizing hormone-releasing hormone

In the first report on the chemical structure of a nonmammalian LH-RH, chicken hypothalamic LH-RH was demonstrated to be [Gln⁸]LH-RH [2–4]. However, these studies and subsequent reports [7,8] did not totally exclude the possibility of a reverse sequence of the two amino acids Leu-Gln. In view of the recently described structure of salmon brain LH-RH as [Trp⁷,Leu⁸]LH-RH [9], we undertook to confirm our earlier conclusion that chicken LH-RH is [Gln⁸]LH-RH and not [Gln⁷,Leu⁸]LH-RH. The immunologic, chromatographic and biological properties of natural chicken hypothalamic LH-RH were compared with those of the two synthetic peptides, [Gln⁸]LH-RH and [Gln⁷,Leu⁸]LH-RH. A radioimmunoassay highly specific for [Gln⁸]LH-RH was developed. Natural chicken LH-RH cross-reacted fully with the antiserum which requires the COOH-terminal Gln⁸ to Gly¹⁰-NH₂ for binding, while [Gln⁷,Leu⁸]LH-RH showed less than 0.1% cross-reaction. On a high resolution reverse phase high performance liquid chromatography system, natural chicken LH-RH co-eluted with [Gln⁸]LH-RH and was well separated from [Gln⁷,Leu⁸]LH-RH. In a chicken anterior pituitary cell bioassay, natural chicken LH-RH and [Gln⁸]LH-RH were equipotent in stimulating luteinizing hormone release, while the relative potency of [Gln⁷,Leu⁸]LH-RH was 4.4%. These data, in particular the use of a specific [Gln⁸]LH-RH antiserum, provide conclusive evidence that chicken LH-RH is [Gln⁸]LH-RH.     

Effect of an antagonistic analog of LH-RH on haloperidol-induced hyperprolactinemia in female rats

The effects of prolonged treatment with the antagonistic analog of LH-RH (N-Ac-D-p-Cl-Phe1,2, D-Trp3,D-Arg6,D-Ala10) LH-RH (ORG 30276) on the hyperprolactinemia induced by haloperidol were investigated in intact or ovariectomized female rats. Treatment with ORG 30276 for 20 days significantly reduced prolactin levels elevated by daily injections of haloperidol in intact as well as in ovariectomized rats. Administration of ORG 30276 also significantly decreased serum LH levels in both types of rats. It is concluded that the LH-RH antagonist ORG 30276 is able to counteract the hyperprolactinemic effect of haloperidol. This effect might be due to a blockade of the action of endogenous LH-RH on the gonadotrophs, which results in a suppressing of the paracrine action of these cells on the lactotroph.     

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.     

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.     

Hydrophobic, aza-glycine analogues of luteinizing hormone-releasing hormone

The effect of combination of the hydrophilic aza-Gly substitution (NHNHCO) at position 10 with hydrophobic, unnatural D-amino acids in position 6 on the potency of luteinizing hormone-releasing hormone (LH-RH) analogues has been investigated. Previously the aza-Gly residue was shown to provide protection from enzymatic cleavage and lead to potency increases in a less hydrophobic series. The compounds were prepared by coupling of the corresponding nonapeptide acids with semicarbazide hydrochloride by the N,N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. The required nonapeptide acids were prepared by the solid phase method on chloromethyl-polystyrene resin using HF/anisole deprotection. The products were purified by preparative reversed-phase high-performance liquid chromatography. The analogues were tested in a rat estrous cyclicity suppression assay designed to show the paradoxical antifertility effects of these compounds. The potencies of [6-(3-benzimidazol-2-yl)-D-alanine), 10-aza-glycine] LH-RH and [6-(3-(5,6-dimethylbenzimidazol-2-yl)-D-alanine), 10-aza-glycine] LH-RH are 40 and 190 times that of LH-RH respectively. The most active compound in this series is [6-(3-(2-naphthyl)-D-alanine), 10-aza-glycine] LH-RH with a potency 230 times that of LH-RH. This compound is 2.3 times as potent as the standard ([D-Trp6, Pro9-NHEt] LH-RH) and appears to be the most potent LH-RH agonist reported.     

Antitumor effects of analogs of LH-RH and somatostatin: Experimental and clinical studies

Many clinical approaches for the treatment of hormone-sensitive tumors are being developed based on analogs of LH-RH and somatostatin. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of LH-RH agonists like [ -Trp⁶]-LH-RH and new LH-RH antagonists free of edematogenic effects such as [Ac- -Nal(2)¹- -Phe(4Cl)²- -Pal(3)³, -Cit⁶, -Ala¹⁰]-LH-RH (SB-75). Agonists and antagonists of LH-RH have been used in patients with prostate cancer and might be also beneficial for the treatment of breast cancer and ovarian, endometrial and pancreatic carcinomas. Some of the effects of LH-RH analogs can be due to direct action since LH-RH receptors have been found in these cancers. The use of sustained delivery systems based on microcapsules of PLG, makes the treatment more efficacious. Octaeptide analogs of somatostatin such as -P s-Trp-NH₂ (RC-160) and related analogs were designed specifically for antitumor activity. These somatostatin analogs, by virtue of having a wide spectrum of activities appear to inhibit various tumors through multiple mechanisms. Direct antiproliferative actions of somatostatin analogs appear to be mediated by specific receptors located on tumor cells. High affinity binding sites for RC-160 and related analogs have been found in human pancreatic, prostate, breast and ovarian cancers and brain tumors such as meningiomas. In vivo administration of analog RC-160 inhibits the growth of Dunning R-3327 prostate cancers in rats, MXT mammary tumors in mice and BOP-induced ductal pancreatic cancers in hamsters. Combination of microcapsules of RC-160 with [ -Trp⁶]-LH-RH results in synergistic potentiation of the inhibition of these cancers. Somatostatin analog RC-160 and LH-RH antagonist SB-75 are the object of further experimental studies and clinical trials aimed at the exploration of their inhibitory effects on the processes of malignant growth.     

Experience with a radioimmunoassay of luteinizing hormone-releasing hormone (LH-RH) in biological material of man.

A radioimmunoassay of LH-RH with a sensitivity of 7.8 pg/ml is described. Labelling and purification techniques, methods for extraction of LH-RH and separation techniques of bound and free labelled hormone are compared. Determination of LH-RH levels in serum after administration of synthetic LH-RH by different routes and measurements of endogenous LH-RH levels in serum of normal subjects and patients with different endocrine diseases as well as in cerebrospinal fluid of normal men are performed. The measurement of exogenously administered LH-RH in serum reflects the disappearance of synthetic LH-RH from peripheral circulation in dependence upon the kind of administration route. The level of endogenous LH-RH was found to be under the limit of the assay in all samples of cerebrospinal fluid and of serum of normal male subjects. The results obtained in the patient groups show that the radioimmunological estimation of endogenous LH-RH in peripheral body fluids does not reflect the hypophysiotrophic role of this hypothalamic peptide.