Action Of Luteinizing Hormone-releasing Hormone Research Articles

NIH conference. Therapeutic applications of luteinizing-hormone-releasing hormone and its analogs.

The chemical structure of luteinizing-hormone-releasing hormone (LHRH) was discovered in 1971 after more than a decade of intensive effort. Subsequent physiologic studies in primates and humans showed that the biologic activity of LHRH depends on the way in which the hormone is administered. Pulsatile administration of LHRH, which mimics the natural secretory pattern, causes sustained secretion of the gonadotrophins. This method of administration has been used to induce ovulation in women with hypothalamic amenorrhea and to induce puberty and spermatogenesis in men with hypogonadotrophic hypogonadism. Continuous infusion, however, produces only transient stimulation of gonadotrophin secretion, followed by a "desensitization" response in which gonadotrophin secretion is inhibited. Thus, LHRH can either augment or inhibit gonadotrophin secretion depending on the mode of administration. Recently, long-acting synthetic analogs of LHRH have been shown to desensitize the pituitary gland and inhibit gonadotrophin release when administered as a single daily subcutaneous injection. These LHRH analogs have proved highly effective in the treatment of prostatic carcinoma and central precocious puberty. They are also being studied as a new approach to contraception and to the treatment of endometriosis and polycystic ovary syndrome.

Interaction between the posterior pituitary and LHRH in the control of LH secretion

We have recently reported that the posterior lobe of the pituitary differentially inhibits the secretion of prolactin (PRL) and luteinizing hormone (LH), but not follicle stimulating hormone (FSH) throughout the estrous cycle. Removal of the posterior pituitary (posterior pituitary lobectomy) results in elevations of plasma LH on all days of the cycle except on diestrus-day-2. In the present study we examined: 1. whether the control of LH release involves an interaction between the posterior pituitary and hypothalamic luteinizing hormone-releasing hormone (LHRH), and 2. whether the elevation of LH seen following posterior lobectomy is due to the removal of a posterior pituitary substance(s) which alters anterior pituitary sensitivity to LHRH. In order to block the action of hypothalamic LHRH, a potent LHRH inhibitory analog (50 μg) was injected SC two hours prior to removal of the posterior pituitary in estrous rats. Administration of the inhibitory analog completely eliminated the elevation of plasma LH seen following posterior lobectomy, but did not alter the posterior lobectomy-induced rise of plasma PRL, or plasma FSH concentrations. In order to test whether anterior pituitary sensitivity to LHRH is altered by posterior lobectomy, a moderate dose of LHRH (15 ng) was administered to both posterior lobectomized and sham lobectomized estrous rats. The time-course and magnitude of the LH response to LHRH was similar in both groups. The results are consistent with the hypothesis that LH secretion is controlled by an interaction between hypothalamic LHRH and the posterior lobe of the pituitary, but this interaction does not appear to involve lobectomy-induced changes in anterior pituitary responsiveness to LHRH. It remains to be determined whether the posterior pituitary contains a substance(s) which is capable of altering hypothalamic release of LHRH.

Ovulation Inhibition in the Pregnant Mare’s Serum Gonadotropin-Treated Immature Rat: A Bioassay for Luteinizing Hormone-Releasing Hormone Antagonists 1

A convenient method for evaluating the biological activity of luteinizing hormone-releasing hormone (LHRH) antagonists was devised. Pregnant mare's serum gonadotropin (PMSG) treatment of immature rats is known to stimulate follicular growth and estrogen production, that in turn stimulates the release of LHRH which triggers an ovulatory discharge of luteinizing hormone (LH) from the pituitary. The present bioassay of the antagonists is based on the inhibition of ovulation in the PMSG-treated rats. Twenty-eight-day-old Sprague Dawley rats maintained under a light period of 12 h/day (lights on at 0630 h) were given 10 IU of PMSG s.c. at 0930 h. On Day 30 of age the antagonist was given s.c. at 1430 h. The rats were killed on the following morning and the oviducts examined for the presence of ova. In addition, the antagonists were compared in their ability to inhibit serum testosterone levels in adult male rats. In the PMSG-treated rats the order of ovulation-inhibiting potency of the following antagonists was: [Ac-D-NAL(2)1,4FD-Phe2,D-Trp3,D-Arg6]-LHRH (LHRH-1) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-NAL(2)3.6]-LHRH (LHRH-2) greater than [Ac-delta 3 Pro1,4FD-Phe2,D-Trp3,6]-LHRH (LHRH-3). The order of potency was confirmed by their antitesticular effects in adult male rats.

LHRH rapidly stimulates phosphatidylinositol metabolism in enriched gonadotrophs

The effects of luteinizing hormone-releasing hormone (LHRH) and human pancreatic growth hormonereleasing factor (hpGRF(l-40)-NH 2) on phospholipid metabolism were studied in rat anterior pituitary cells in primary culture. In a 4-fold enriched population of gonadotrophs, 30 nM LHRH increased 32P; incorporation into phosphatidic acid (PA) as early as l min after its addition. Phosphatidylinositol (PI) labeling was increased l min later. The stimulatory action of LHRH was observed in both phospholipids up to 100 min, the last time interval studied. The decapeptide did not affect 32P i labeling of phosphatidylcholine (PC), lysoPC, phosphatidylethanolamine or phosphatidylserine. Dose-response studies performed after 25 min of incubation showed an ED 50 value of LHRH action at approximately 1 nM for PI labeling. In contrast, the addition of 0.1 μM GRF to anterior pituitary cells enhanced 32P i incorporation only into PC after a 60 min incubation period. The present data suggest that stimulation of acidic phospholipid metabolism, particularly an increase in PA-PI turnover, may represent an early event in the mechanism of action of LHRH but not GRF in the anterior pituitary gland.

Long-Term Stability of Aqueous Solutions of Luteinizing Hormone-Releasing Hormone Assessed by an In Vitro Bioassay and Liquid Chromatography

The stability of aqueous solutions of luteinizing hormone-releasing hormone (LHRH) after extended storage at various temperatures was investigated using a newly developed HPLC assay and an in vitro dispersed pituitary cell culture bioassay. Good correlations were obtained between the potency obtained by HPLC and bioassay in samples stored at 37°C or subjected to different stress conditions. No significant decrease in activity of LHRH was observed in aqueous solutions stored at 37°C for up to 10 weeks, at 4°C for 2 years, or subjected to repeated freezing and thawing for 5 d. Heating to 60°C in sterile pH 9.0 buffer up to 11 d and storage at ambient temperature in nonsterile solution for 4 months produced well-distinguished degradation products and a decrease in potency. It is concluded that sterile aqueous solutions of LHRH are stable for at least 10 weeks at 37°C and, thus, could be reliably used for chronic administration when long-term stability at body temperature is important.

Pharmacokinetics and metabolism of lhrh agonists, clinical aspects

The authors investigated the pharmacokinetic measurement methods for determination of the the highly active luteinizing hormone-releasing hormone (LHRH) agonist buserelin in animal plasma and serum with emphasis on the detection of intact peptide and metabolities. These methods were applied to the study of endometriosis prostate cancer and precocoious puberty and to the monitoring of use of a buserelin nasal spray as a contraceptive. Even after a small nasal spray dose (400 mcg) the biologic effect of buserelin on LH release was striking and unequivocal. Although the plasma LH increase was measured for over 6 hours the plasma level of buserelin was only transiently elevated. Both the acute LH release and the urinary excretion of immunoreactive buserelin appear to be the most reliable pharmacokinetic parameters during low-dose stimulation. The utility of buserelin determination in unextracted urine was confirmed by application of the method to dose-finding studies. Parameters used for calculations is studies on urinary excretion of immunoreactive buserelin after nasal spray administration included urinary buserelin excretion buserelin plasma level and the LH release induced. Due to the high doses used in pituitary-gonadal suppression by LHRH agonists as well as the sustained levels observed during high-dose injection and longterm infusion therapy the risk of antibody formation must be evaluated carefully. No formation of antibodies was detected in any of the clinical studies. This low antigenic potential is considered a result of the small moelecular size of LHRH agonists and the rapid rate of degradation by exo- and endopeptidases. In general determination of urinary excretion is the preferred method for therapy control although determination of plasma levels can be performed for rapid evaluation or additional confirmation.

Luteinizing hormone-releasing hormone increases dopamine turnover in the lateral palisade zone of the median eminence and reduces noradrenaline turnover in the nuc. Preopticus medialis of the hypophysectomized male rat

By means of catecholamine fluorescence histochemistry in combination with quantitative microfluorimetry the effects of luteinizing hormone-releasing hormone (LH-RH) has been studied on the amine levels and turnover in discrete catecholamine (CA) nerve terminal populations in the hypothalamus of the hypophysectomized male rat. LH-RH highly selectively increases dopamine (DA) turnover in the lateral palisade zone of the median eminence and highly selectively reduces noradrenaline turnover in the nucleus preopticus medialis without influencing amine levels in these CA nerve terminal systems. It is suggested that the ultrashort feed-back action of LH-RH involves increased release of DA in the lateral palisade zone leading to activation of the DA receptor inhibiting LH-RH release as well as a reduced noradrenaline release in the nucleus preopticus medialis leading to a reduced excitatory influence on the LH-RH neurons at the soma-dendritic level.

Luteinizing Hormone-Releasing Hormone (LHRH) and its analogs for contraception in women: A review

In animals, LHRH agonists have multiple sites of action including the pituitary, the gonads, and the reproductive tract. In humans, the major antifertility action of this class of peptides is believed to be mediated via the pituitary. Studies in women have indicated that potent LHRH agonists can block ovulation when administered once daily. In the volunteers who have used these agents no serious side effects were observed, although some women experienced irregular bleeding or amenorrhea. It is anticipated that formal clinical trials could be conducted in the near future to determine the efficacy of continuous LHRH agonist administration. Early attempts to use an LHRH agonist to produce luteal insufficiency, luteolysis, or interruption of pregnancy have either been unsuccessful or the results are still too preliminary to ascertain whether these approaches warrant further trials. LHRH antagonists are believed to act by inhibiting the action of LHRH on the pituitary. Although some of these peptides are known to be active in women, very large doses have been required. Recently several investigators have produced LHRH antagonists with increased potency. In the near future, it should be possible to determine whether these peptides should be considered as potential contraceptives in men or in women.

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [ D-Phe 6,desGly 10]-LHRH ethylamide and [ D-Ser(Bu t) 6,desGly 10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al 2O 3 catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [ D-Phe 6,desGly 10]- and [ D-Ser(Bu t) 6, desGly 10]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.

Evolution of Neurohormonal Regulation of Reproduction in Lower Vertebrates

Immunological and chromatographic studies demonstrate that the gonadotropin (GtH) releasing hormone (GnRH) in the brain of chondrichthyes, teleosts, reptiles and birds is different from luteinizing hormone-releasing hormone (LH-RH) of mammals. LH-RH is present in brain extracts from amphibia. The alterations in the structure of LH-RH found in various of the non-mammalian vertebrates are at positions 7 and 8; the structure of chum salmon GnRH is Trp7-Leu8-LH-RH, and in chickens is Gln8-LH-RH. A response to injection of LH-RH or its agonistic analogues, in terms of increased blood levels of GtH or a gonadal response such asovulation indicative of increased GtH secretion, has been found in all classes of vertebrates, and the cephalochordate amphioxus. This suggests a basic similaritiy of the GnRH receptors throughout vertebrates, and that the ancestral origin of the system was in the invertebrate chordates. Immunohistochemical studies demonstrate that many lower vertebrates have perikarya containing LH-RH-like material in the preoptic and ventrobasal hypothalamic regions. Brain lesioning studies provide functional evidence for GnRH from both locations in amphibia and only the ventrobasal hypothalamus in teleosts. Brain lesioning studies on goldfish suggest the presence of a GtH release-inhibitory factor (GRIF). Dopamine has GRIF activity in goldfish and common carp to modulate the actions of LH-RH and spontaneous release of GtH. How widespread this system for dual neurohormonal regulation of GtH secretion is in vertebrates is not known.

Action of luteinizing hormone-releasing hormone: involvement of novel arachidonic acid metabolites.

Anterior pituitary cells were incubated in the presence of luteinizing hormone-releasing hormone and one of three inhibitors of arachidonic acid metabolism:indomethacin, an inhibitor of the cyclooxygenase system; nordihydroguaiaretic acid, an antioxidant that inhibits lipoxygenase; and icosatetraynoic acid, an acetylenic analogue of arachidonic acid that blocks all known pathways of arachidonic acid metabolism. Indomethacin was ineffective in blocking luteinizing hormone-releasing hormone-stimulated luteinizing hormone secretion. Nordihydroguaiaretic acid was only marginally capable of inhibiting luteinizing hormone-releasing hormone-stimulated luteinizing hormone secretion. Icosatetraynoic acid at 10 microM completely inhibited stimulated luteinizing hormone secretion. Addition of several epoxygenated arachidonic acid metabolites to cells in vitro resulted in secretion of luteinizing hormone equal to or greater than that induced by 10 nM luteinizing hormone-releasing hormone. The half-maximal effective dose for these compounds was approximately 50 nM. The 5,6-epoxyicosatrienoic acid was the most potent of the compounds tested. These studies suggest that luteinizing hormone-releasing hormone-stimulated luteinizing hormone release is closely coupled with the production of oxidized arachidonic acid metabolites. Moreover, one or more of the epoxygenated arachidonic acid metabolites might be a component of the cascade of reactions initiated by luteinizing hormone-releasing hormone that ultimately results in secretion of luteinizing hormone.

Stimulation of phosphatidic acid and phosphatidylinositol labeling in luteal cells by luteinizing hormone releasing hormone.

Luteinizing hormone-releasing hormone (LHRH) causes a rapid and marked increase of [32P]orthophosphate incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The neurohormone exerts its stimulatory effect at an ED50 value of approximately 15 nM. Human chorionic gonadotropin (hCG) has no effect alone and does not interfere with the LHRH-induced PA-PI labeling. The rapidity and the specificity of the effect of LHRH suggest that the stimulation of the PA-PI cycle may well serve as a potent transducing mechanism responsible for the direct action of LHRH and its agonists at the ovarian level.

The rapid effects of Luteinizing hormone releasing hormone agonists and antagonists on the mouse pituitary IN VITRO

The effect of luteinizing hormone releasing hormone (LHRH) and its analogs on the release of FSH and LH by 20 day old whole mouse pituitary incubated in vitro for 3–4 hrs was investigated. Three agonistic analogs (AY 25650, 25205 and Buserelin) all of which are reported to be superactive in vitro showed approximately the same potency in this in vitro test system. Preincubation of the pituitaries for 1 h with the antagonistic analogs [Ac Dp C1 Phe 1,2, D Trp 3, D Phe 6, D Ala 10] LHRH and [Ac Dp C1 Phe 1,2, D Trp 3, D Arg 6, D Ala 10] LHRH inhibited the secretion of LH and FSH induced by 2.5 × 10 −9M LHRH. The inhibitory response was dose dependent. The continued presence of the antagonists was not required for effective suppression of the LHRH effect. Experiments designed to find out the minimum time required for eliciting suppression of LHRH revealed that preincubation of the pituitary with the second antagonist for 5 mins followed by removal was adequate to produce effective inhibition of gonadotropin release. At lower doses of the antagonists,LH release was more effectively inhibited than FSH release. The results suggest that antagonistic analogs can effectively bind to LHRH receptors in the whole pituitary incubation preventing the subsequent action of LHRH. With the present incubation system assessment of bioactive LH and FSH release is possible within 24 hrs.

Luteinizing hormone releasing hormone prevents testicular atrophy in golden hamsters exposed to a short photoperiod: temporal difference in effectiveness of administration of luteinizing hormone releasing hormone.

Exposure of male golden hamsters to short photoperiods of 6 h light: 18 h darkness led to testicular and accessory sex organ atrophy in 5 weeks. Short photoperiods also significantly depressed serum levels of LH, FSH, prolactin and testosterone in samples obtained by decapitation, but not in samples collected on the preceding day under ether anaesthesia. Injections of luteinizing hormone releasing hormone (LH-RH) at 09.00 h (lights on) or at 15.00 h (lights off) prevented testicular regression when compared with hamsters receiving injection vehicle only. However, the hamsters receiving LH-RH injections at lights on had significantly greater testicular weight and accessory sex organ (seminal vesicles and coagulating glands) weight and testosterone concentration than those receiving LH-RH at lights off. No increase in testicular weight was observed in hypophysectomized male hamsters given the same LH-RH injections and the same lighting regimen. These results indicate that LH-RH alone can prevent, at least partially, testicular and sex organ atrophy and increase serum testosterone concentration by stimulating release of LH and FSH in hamsters exposed to short photoperiods, involving temporal difference of LH-RH action. Further implications of the results are discussed.

The regulation of LHRH action at the pituitary and gonadal receptor level: A review

Luteinizing hormone releasing-hormone (LHRH) and its highly active agonists are under clinical investigation for the control of reproductive function and for suppression of hormone dependent tumours. The regulation of LHRH action by pituitary receptors and expression of the biological LHRH effect by gonadotropin release and activation of steroid biosynthesis are discussed in this context. Pituitary LHRH receptors are controlled by autoregulation via endogenous LHRH secretion. The gonadal response to LHRH stimulation is regulated by LH action on receptors for LH, prolactin and FSH. Pituitary and gonadal inhibition are achieved by different mechanisms. Continuous exposure to LHRH blocks gonadotropin release and reduces pituitary LH/FSH content, whereas inhibition of steroid biosynthesis requires daily LH release to maintain receptor down-regulation. Pituitary enzymes involved in LHRH degradation at the receptor site are required for terminating hormone action, but their role in modulating hormonal responsiveness is secondary to receptor regulation. Direct gonadal effects of LHRH are exerted in the presence of gonadotropins by modulating the gonadotropin effect, e.g. in hypophysectomized animals. The presence of specific receptors for LHRH agonists in ovarian and testicular tissue suggests local control mechanisms for gonadotropin activation of steroid biosynthesis.

Differential effect of althesin versus chloralose-urethane anesthesia on in vivo LHRH and LH release in the proestrous rat.

Anesthetic doses of Althesin and chloralose-urethane were compared as to their effects on neuroendocrine reproductive processes in diestrous and proestrous rats. Proestrous rats treated with Althesin experienced a 5-fold increase in immuno-reactive luteinizing hormone releasing hormone (LHRH) concentration in methanolic extracts of pituitary stalk plasma. This LHRH increase was 67% greater than that of proestrous rats administered chloralose-urethane. Moreover, Althesin given before the onset of the critical period did not suppress the proestrous surge of LHRH in stalk plasma. The Duncan's multiple range test indicated stalk plasma LHRH activity was significantly elevated in proestrous rats anesthetized with Althesin (p less than 0.05) but not with chloralose-urethane. However, the luteinizing hormone (LH) concentration in peripheral plasma increased 14 to 18-fold in all proestrous rats, including those anesthetized with chloralose-urethane. Evidently, in chloralose-urethane-treated rats, the smaller LHRH surge during proestrus is sufficient to elicit maximum release of LH from the anterior pituitary gland. This suggest that under normal conditions the amount of LHRH secreted into portal blood during proestrus is approximately 2-fold greater than that necessary to elicit maximum secretion of LH. The results also show that picogram concentrations of LH in the stalk plasma will stimulate the release of nanogram quantities of LH into the peripheral plasma of the same rats. Moreover, the sensitivity of the pituitary gland to LHRH stimulation via the portal circulation increases nearly 3-fold between diestrus and proestrus.

Endocrine and behavioral effects of continuous exposure of male rats to a potent luteinizing hormone-releasing hormone (LHRH) agonist: evidence for central nervous system actions of LHRH.

Continuous exposure of intact male rats to [6-D-(2-naphthyl)-alanine]LHRH, a highly potent LHRH agonist, diminished testis and pituitary weights and decreased basal plasma levels of testosterone and LH. In addition, treatment with this analog abolished the pituitary hypertrophy and LH hypersecretion normally associated with castration. Alterations of male sex behavior were also noted in these animals. Analog-treated intact rats showed longer intromission latencies and mount latencies in early tests and ceased to show sexual behavior 6-8 weeks after initiation of treatment. In addition, the gradual decline in behavior normally observed after castration was markedly accelerated by analog administration. Castrates bearing 5-mm Silastic testosterone-containing capsules which maintained copulatory performance displayed behavioral deficiencies when given the peptide. No significant effect of analog was noted in rats with 30-mm Silastic implants, which provide a higher level of testosterone replacement. Continuous analog treatment promotes endocrine effects consistent with complete antagonism of the effects of endogenous LHRH. The behavioral disruption associated with this manipulation suggests a role for endogenous LHRH in maintaining sex behavior in a low testosterone environment.

Some analogues of luteinizing hormone-releasing hormone with substituents in position 10.

As part of our studies on the design of agonists of the luteinizing hormone-releasing hormone (LH-RH), we have synthesized the [des-Gly-NH2(10)]-LH-RH N-methylhydrazide (1), the corresponding thiosemicarbazide (2), and the N-formyl- (3) N-acetyl- (4) and N-(trifluoroacetyl)hydrazide (5). Analogue 1 may be regarded as isosteric with [des-Gly-NH2(10)]-LH-RH N-alkylamides which are, in general, potent agonists. Analogues 2-5 may be regarded as isosteric with [aza-Gly-NH2(10)]-OH-RH, which is equipotent with the hormone. The required protected intermediates were prepared by solid-phase synthesis, and the free peptides were prepared from them by deprotection with HF, followed by purification on Sephadex G-25. Bioassay of these analogues with rat hemipituitaries in vitro showed the following values as percentages of the hormonal values for the release of LH and FSH respectively: N-methylhydrazide (1), 17 and 11%; semithiocarbazide (2), 6.5 and 4.6%; N-formylhydrazide (3), 15.3 and 10%; N-acetylhydrazide (4), 1.2 and 0.6%; N-(trifluoroacetyl)hydrazide (5), 1.0 and 0.9%. Thus, these types of isosteric substitutions are inimical to the preservation of the high biological activity of LH-RH.

Pressor action of luteinizing hormone-releasing hormone in the rat.

Pressor action of luteinizing hormone-releasing hormone in the rat.

Cellular mechanism of the angtigonadotropic action of luteinizing hormone-releasing hormone in the corpus luteum.

In incubations of rat luteal cells, LHRH or an agonistic analog (D-Trp6)LHRH showed a dose-response inhibition of LH-stimulated cAMP accumulation and progesterone secretion. The dose of LHRH estimated to produce 50% inhibition of LH-dependent cAMP accumulation was 3 × 10-7 M, and the LHRH dose estimated to inhibit progesterone secretion by 50% was 8 × 10-6 M. (D-Trp6)LHRH was 4- and 40-fold more potent than LHRH on LH-dependent cAMP accumulation and progesterone secretion, respectively. Although LHRH (8 × 10-6 M) inhibited LH-stimulated cAMP accumulation at 100 and 1000 ng/ml LH, inhibition of progesterone secretion by LHRH was evident at 100 ng/ml, but not at 1000 ng/ml LH. Thus, high levels of gonadotropin may overide the inhibitory effect of LHRH on progesterone secretion. LHRH did not inhibit the stimulation of progesterone secretion induced by dibutyryl cAMP or a combination of dibutyryl cAMP and LH (100 ng/ ml). However, LHRH inhibited LH-dependent cAMP accumulation in both the absence and presence ...