Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using ¹²⁵I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu^t)⁶]LHRH(1–9) ethylamide and an antagonist DpGlu¹, DPhe², DTrp^3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K_a values for the agonist, LHRH and the antagonist were 2.1 × 10⁹M^?1, 0.92 × 10⁸M^?1 and 0.76 × 10⁹M^?1, respectively, and the binding capacity was 116 fmoles/10⁶ pituitary cells.     

Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

Binding of native, glucosamine-labeled, luteinizing hormone to Leydig cells.

A method for obtaining pituitary glycoprotein hormones labeled either in the protein backbone or in the carbohydrate moiety, by incubation of pituitary slices in the presence of radioactive leucine or glucosamine, has been developed. This report describes the subsequent purification of the “native” labeled luteinizing hormone from the incubation medium and its use in binding studies with testicular tissue. The purified radioactive luteinizing hormone specifically bound to Leydig cells could be displaced with excess human chorionic gonadotropin, but was unaffected by excess follicle stimulating hormone. Binding data indicated that $\text{K}{}_{\mathrm{d}}\text{= 5.2 × 10}{}^{\mathrm{?9}}\text{m}$ and approximately 6 × 10⁴ binding sites per cell.     

The release of pituitary gonadotrophins by luteinizing hormone releasing hormone in the intact,castrated and aspermatogenic rat

The change in serum gonadotrophin concentration in response to synthetic Luteinizing Hormone Releasing Hormone (LHRH - 400 ng i.v.) was investigated under barbiturate anaesthesia in adult male rats either chronically castrated, rendered aspermatogenic by the administration of α-chlorohydrin 12–16 weeks previously (to remove inhibin), or treated with vehicle. A single injection of LHRH increased serum LH and FSH concentrations similarly in both intact and aspermatogenic rats. In castrated rats the amount of LH released was much greater and the FSH secretion sustained. A second injection produced a similar increase although a second peak of FSH could not be detected in castrated rats as the FSH level was still elevated. The increase in LH levels was two to three times larger in response to the second injection of LHRH than to the first in all groups. The results do not support the hypothesis that the enhanced gonadotropin response to castration in the aspermatogenic rat is due to increased pituitary sensitivity to LHRH.     

Catecholamine stimulation of androgen production by rat Leydig cells. Interactions with luteinizing hormone and luteinizing hormone-releasing hormone

The mechanism(s) of the development of response to catecholamines (CA) by Leydig cells in culture was investigated with the use of primary culture of purified Leydig cells of adult rats. The interactions of a CA agonist, isoproterenol (ISOP), with luteinizing hormone (LH) and a luteinizing hormone-releasing hormone agonist analog (LHRHa) on production of androgen by the Leydig cells were also studied. Cells incubated with ISOP for 3 h increased release of cyclic adenosine 3',5'-monophosphate (cAMP) to similar extents at 0, 3, and 24 h of culture. The beta-agonist did not increase androgen release at 0 h but had a concentration-dependent effect at 3, 24, and 48 h of culture, with maximal effects at 24 h. LH stimulated high increases in production of cAMP and androgen by the cells at 0-24 h of culture. Leydig cell beta-receptors decreased with culture time. Low concentrations but not high levels of LH had additive effects with ISOP on androgen release. ISOP showed a complex interaction with LHRHa on androgen release. Chronic exposure of Leydig cells to LHRHa reduced basal androgen release as well as release of androgen stimulated by ISOP, forskolin, and LH. These studies suggest that the development of response to CA by rat Leydig cells is a postreceptor, postcAMP event and showed that CA can interact with LH or LHRH to regulate Leydig cell function.     

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.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary and ovarian responses to luteinizing hormone releasing hormone in a rat with polycystic ovaries

Female rats injected with a single dose of 2 mg estradiol valerate (EV) develop anovulatory acyclicity characterized by persistent vaginal cornification and the formation of multiple large cystic follicles on the ovaries. In order to determine if these effects of EV are accompanied by changes in ovarian and/or pituitary function, the following studies were conducted. Ovarian androgen production was determined by the measurement at 4, 5 and 6 weeks after EV treatment of circulating dehydroepiandrosterone, androstenedione and testosterone. The capacity of the polycystic ovary to ovulate in response to luteinizing hormone releasing hormone (LHRH) stimulus was assessed. Ovarian histology was examined at the termination of the study (9 weeks after EV treatment). Pituitary function was assessed 9 weeks after the EV treatment by examining the acute changes in plasma luteinizing hormone (LH) concentration in response to a double pulse of LHRH. Plasma concentrations of the androgens were unchanged over the 3-week sampling period and were similar to those found in sesame-oil-treated normal cycling control rats. The ovaries from EV-treated animals were smaller than those of controls and the cystic follicles exhibited marked thecal hypertrophy and attenuation of the granulosa cell layer. The basal plasma LH concentration at 9 weeks after EV treatment were significantly lower than in proestrus controls and plasma concentrations of LH elicited by LHRH pulses was significantly lower than in controls. The relative increase in plasma LH following the LHRH stimulus was, however, greater in the EV-treated animals than in controls. In spite of the diminished LH surge elicited in response to LHRH, the EV-treated animals ovulated as indicated by the presence of fresh corpora lutea on the ovaries. These results indicate that androgens are not responsible for the polycystic ovarian condition in this system and that the polycystic ovary is capable of ovulatory function when appropriately stimulated.     

Binding of luteinizing hormone releasing hormone to human serum proteins--influence of a chronic treatment with a more potent analogue of LH-RH.

Binding of 125I-LH-RH and its analogue, 125I-6-D-Leu-10-Des-Gly-Ethylamide-LH-RH (6-D-LH-RH) in male serum was studied in 10 healthy males and in 11 patients with idiopathic gonadotropin deficiency (IGD) before and during treatment with 6-D-LH-RH. Using either equilibrium dialysis (A) or ethanol precipitation (B) 13.57 +/- 0.69% (A) or 19.32 +/- 1.73% (B) of LH-RH and 7.12 +/- 0.86% (A) or 14.56 +/- 1.06% (B) of the analogue were in the bound form, without difference between normal subjects and IGD. Capacity of this binding was high (greater than 9 less than 18 mu-Mol LH-RH/0.06 mMol of protein), affinity very low, and the binding almost completely disappeared following removal of albumins by affinity chromatography. Chronic treatment with 6-D-LH-RH did not alter these binding characteristics. These observations suggest non specific albumin binding of LH-RH in male serum and stress the role of this decapeptide as a rapid modulating regulator of gonadotropin secreting system.