Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation

The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.     

Antagonists of the luteinizing hormone releasing hormone (LHRH) with emphasis on the TRP7 of the salmon and chicken II LHRH's

The sequences of four naturally occurring luteinizing hormone releasing hormones (LHRH's) differ only in positions 5, 7 and 8. Salmon and chicken II LHRH's have Trp7; porcine/ovine (P/O) and chicken I LHRH's have Leu7. The receptor for P/O LHRH might effectively bind certain antagonists with Trp7. Thirteen antagonists having Trp7 and eight antagonists with other substitutions in position 7 were synthesized. One of the thirteen antagonists with the natural Trp7, [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3,D-Arg6,Trp7,D- Ala10]-LHRH, not only maintained activity, but had increased potency (ca. 58%; 90% antiovulatory activity/250 ng; rats) in comparison with the companion analog with the natural Leu7 of P/O LHRH. The other twelve Trp7-antagonists had lower potency.     

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.     

Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells

The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.     

Luteinizing hormone secretion from the quail pituitary in vitro

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.     

Comparative studies on the hypotensive effect of LHRH antagonists in anesthetized rats

Certain neuropeptides are known to cause a hypotensive response, thought to be due to mast cell degranulation. The effects of five antagonists of luteinizing hormone-releasing hormone on blood pressure and heart rate were compared in the anesthetized rat. When given intravenously, all five compounds induced hypotensive and bradycardiac effects. The order of potency for these effects was Nal-Arg Antagonist approximately detirelix [( N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10]LHRH) greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,D-hArg(Et2)6,L-hArg (Et2)8,D-Ala10]LHRH (RS-26306) approximately antide greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,6, L-hArg(Et2)8,D-Ala10]LHRH (RS-15378) and did not parallel the order of antiovulatory potencies of these compounds. The hypotensive activity of LHRH antagonists, therefore, appeared dissociable from their antiovulatory activity. RS-26306 and RS-15378 appeared to have the greatest therapeutic ratios.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.     

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

A comparison of PMSG and LHRH agonist treatment in the induction of ovulation in the anoestrous ewe

The aim of this experiment was to compare the use of pregnant mares' serum gonadotrophin (PMSG) with that of a luteinizing hormone releasing hormone (LHRH) agonist in the induction of ovulation in anoestrous sheep. Anoestrous ewes were treated with progestagen-impregnated sponges for 12 days. They were given either PMSG at the time of sponge withdrawal or the LHRH agonist D-Ser(But)⁶desGlyNH₂¹⁰LHRH ethylamide 20 h after sponge withdrawal. This protocol was followed over 2 consecutive years. Plasma concentrations of oestradiol and LH were measured, and in the first year a comparison was made of the ovulation rate, conception rate and luteal function of the two groups after artificial insemination. During the first year, all of the PMSG-treated group but none of the agonist-treated group exhibited oestrus. Five of the eight PMSG-treated ewes had embryos in utero at slaughter whilst none was present in the agonist-treated ewes. The secretion of progesterone was greatest in the PMSG-treated ewes (P < 0.001). During the second year, a more frequent blood-sampling regime was employed. Increased plasma concentrations of LH occurred within 3 h of agonist administration. Plasma oestradiol concentrations peaked at 20 h and 45 h after sponge withdrawal in both groups. Both peaks were larger in the agonist-treated group. It is concluded that a single dose of the highly potent LHRH agonist is unable to produce normal luteal function or conception using the present protocol.     

The luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, has no direct effect on spermatogenesis in the rat

Treatment of intact rats with luteinizing hormone-releasing hormone (LHRH) agonists has been shown to produce atrophy of a variable number of testicular seminiferous tubules. These findings raised the question of a possible direct versus indirect action of LHRH agonists on spermatogenesis. To answer this question, we treated hypophysectomized rats with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, dihydrotestosterone (DHT), or a combination of these two compounds for a period of 1 mo. Treatment of hypophysectomized animals with the LHRH agonist alone had no significant effect on the atrophy of seminiferous tubules found after hypophysectomy. DHT, however, maintained spermatogenesis at 80% of the level seen in intact animals. When DHT and the LHRH agonist were administered in combination, the stimulatory effects of DHT were observed with no significant interference caused by the LHRH agonist. This study shows that an LHRH agonist has no direct effect on the morphology of the seminiferous tubules in the absence of the pituitary gland and strongly suggests that the atrophy observed in the testis after LHRH agonist treatment in intact animals is mediated by the LHRH agonist-induced changes in luteinizing hormone secretion and/or direct action of the peptide on Leydig cells.     

Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

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-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various tissues 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-Phe6,desGly10]- and [D-Ser(But)6,desGly10]-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 the analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.     

Luteinizing hormone releasing hormone mediates naloxone's effects on serum luteinizing hormone levels in normal and morphine-sensitized male rats

Naloxone produces large increases in serum luteinizing hormone (LH) levels in normal males and females, supporting a role for endogenous opioids (EOP) in the tonic inhibition of LH. Since the antagonist apparently exerts no important effects on the pituitary, the reasonable assumption has been made that it elevates gonadotropin levels by affecting the release of LH-releasing hormone (LHRH) from the hypothalamus. However, at present there is no direct in vivo evidence supporting this widely-held view. In an attempt to directly demonstrate that naloxone increases the secretion of LHRH, and thereby elevates serum LH levels, we examined whether a potent synthetic antagonist of LHRH ( [D-p Glu1, D-Phe2, D-Trp3,6]-LHRH, GPT-LHRH) blocked the effects of naloxone in male rats with a normal response to naloxone and in those with a markedly enhanced sensitivity to the drug induced by a brief period of morphine pellet implantation. Our results demonstrated that GT-LHRH antagonized equipotent doses of LHRH (100 ng/kg) and naloxone (0.5 mg/kg) over a similar time course with approximately the same AD50. Most importantly, however, we showed that the GPT-LHRH produced equivalent, parallel shifts to the right in the dose-response curves for LHRH and naloxone, indicative of competitive inhibition. We also found that GPT-LHRH completely abolished the enhanced response to naloxone's effects on LH which occurs in morphine-pretreated rats. Since we observed no competition between LHRH and naloxone for their binding sites in pituitary or brain, the only viable interpretation of our results is that naloxone increases LH by inducing the release of LHRH.     

半胱胺对草鱼下丘脑-脑垂体组织共孵育中生长激素分泌的影响

采用静态孵育和放射免疫测定技术,研究了生长抑素抑制剂半胱胺盐酸盐对草鱼脑垂体组织单独孵育或下丘脑脑垂体组织共孵育中生长激素分泌的影响。结果表明：脑垂体组织单独孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌无影响;而下丘脑脑垂体组织共孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌有明显影响,且是剂量依存的。神经肽hGHRH、sGnRH—A和LHRH—A对CSH影响的下丘脑脑垂体组织共孵育中生长激素分泌均无协同作用。我们认为,半胱胺盐酸盐可在下丘脑水平调节生长激素释放,半胱胺盐酸盐调节草鱼离体生长激素分泌是由下丘脑途径介导的。     

On the importance of position one of ovulation inhibitors, as based on studies on [D-Phe2, Pro3, D-Phe6]-LHRH.

Substitution of cyclopentylcarbonyl-(Cpc) for 1 in the effective and potent antiovulatory inhibitor, [D-Phe², Pro³, D-Phe⁶]-LHRH (I) retained the $\text{in vitro}$ potency. We know of no other inhibitor of the luteinizing hormone releasing hormone (LHRH) with a modification at position 1, which is as potent $\text{in vitro}$. This result agrees with the concept of the role of 1, D-Phe², Pro³, D-Phe⁶]-LHRH did not inhibit ovulation in rats at the same dosage as did I; this result is under study to circumvent. Des-Gly¹⁰-[D-Phe², Pro³, D-Phe⁶]-LHRH ethylamide and [Glu¹, D-Phe², Pro³, D-Phe⁶]-LHRH were significantly less active $\text{in vitro}$ than I.     

Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo. A histological and immunohistochemical study

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapasules of a biodegradable polymer poly(DL-lactide-co-glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [D-Trp6]-LHRH and microcapsules of the LHRH antagonist SB-75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue-compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [D-Trp6]-LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [D-Trp6]-LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.     

Design, synthesis and bioassays of antagonists of LHRH which have high antiovulatory activity and release negligible histamine

Potent antagonists of the luteinizing hormone releasing hormone have been achieved which release negligible histamine. [N-Ac-D-2-Nal1, D-pClPhe2, D-3-Pal3, NicLys5, D-NicLys6, ILys8, D-Ala10]-LHRH showed 100%AOA/1 microgram and 36%/0. 5 micrograms; ED50 greater than 300. [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3, PicLys5, D-PicLys6, ILys8, D-Ala10]-LHRH showed 100% AOA/0.5 micrograms and 40%/0.25 micrograms; ED50, 93 +/- 11, and is the most potent of 52 new peptides. These antagonists feature designs with weakly basic acylated D-Lys6, notably D-NicLys6 and D-PicLys6 and alkylated Lys8 or Orn8, e.g., ILys8 and IOrn8, and NicLys5 and PicLys5. Concepts included balanced overall basicity, superiority of ILys8 and IOrn8 which are sequence dependent and sensitivity of positions 5 and 6 for potency.     

Regulation of follicular development by diethylstilboestrol in ovaries of immature rats

Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

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-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ 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-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-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.