Properties of a potent LHRH antagonist (Org 30850) in female and male rats.

Org 30850 (Ac-D-pClPhe1,2,D-Bal3,D-Lys6,D-Ala10-LHRH) is a novel LHRH antagonist, which is being developed for the treatment of hormone-dependent disorders. The activities of this compound with respect to its endocrinological properties and side-effects were tested in rats and the results were compared with one of the first LHRH antagonists: Ac-D-pClPhe1,2,D-Trp3,D-Arg6,D-Ala10-LHRH (Org 30276). A single subcutaneous (s.c.) dose of 0.3 micrograms/kg Org 30850 administered to rats in pro-estrus gave inhibition of ovulation in approx. 50% of the rats, whereas Org 30276 was approx. 4 times less potent. The effect of a single s.c. injection of Org 30850 on testosterone levels in young adult male rats was also studied. The administration of 250 micrograms/kg or higher of Org 30850 induced a significant decrease in testosterone levels after 3 h, this effect lasted for at least 48 h. Treatment of female rats for 14 days with a daily dose of 12 micrograms/kg Org 30850 decreased statistically significantly uterine and ovarian weights. At a daily dose of 50 micrograms/kg Org 30850 completely suppressed estrous cycles and significantly decreased estradiol and FSH serum levels. The LH levels were below the detection level in both control and treated animals on the (expected) second day of di-estrus. Treatment of male rats for 14 days (25-200 micrograms/kg) resulted in a dose-dependent reduction of the gonads, accessory sex organs, testosterone levels and gonadotrophins. The decrease in gonadal function in both sexes was reversible since the females proved to be as fertile as the controls 6 weeks after the last treatment and an almost complete recovery of the weight of testes, seminal vesicles and ventral prostate was observed in the males 4 weeks after cessation of treatment. In contrast to Org 30276, Org 30850 exerted very slight irritation at the site of injection and no edematous reactions in the extremities at a daily dose of up to 8 mg/kg in male rats. It is concluded that Org 30850 is a very potent LHRH antagonist without edematous reactions and with a more favourable therapeutic index than Org 30276.     

Evaluation of the anaphylactoid activity of a new LHRH antagonist

ORF 23541 [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,Ser4,Nic-Lys5,D-Nic-Lys 6, Leu7, I-Lys8, Pro9,D-Ala10NH2; "Nal-Lys antagonist"] was identified as a potent LHRH antagonist without significant anaphylactoid activity. It blocked ovulation in proestrus rats when administered subcutaneously with an ED50 of 5.8 micrograms/kg. Much higher doses of ORF 23541 than of other antagonists were required to induce a cutaneous anaphylactoid-like reaction. Intradermal administration of ORF 23541 caused an 8.75 x 8.75 mm wheal response with estimated doses of 10.9 and 13.7 micrograms in rats and guinea pigs, respectively. These doses were at least 10 times greater than that required of other LHRH antagonists for the same response. ORF 23541 also did not alter pulmonary function in guinea pigs or dogs when administered intravenously at doses up to 10 mg. These results indicate that ORF 23541 represents a new generation of LHRH antagonists with an improved safety margin.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.     

Postcastration rise in plasma gonadotropins is blocked by a luteinizing hormone-releasing hormone antagonist

The dependence of the acute increases in plasma gonadotropins following castration on luteinizing hormone-releasing hormone (LHRH) was assessed with the use of a potent LHRH antagonist [ALHRH; (Nac-L-Ala1,p-Cl-D-Phe2,D-Trp3,6) LHRH]. Blood samples were collected from male and female rats at the time of castration and 2, 4, 8, 12, 24 and 48 h following and plasma gonadotropin levels were determined. Immediately following castration (diestrus I for females) animals received one of the following treatments: females-vehicle, 100 micrograms ALHRH, 50 micrograms estrogen benzoate (EB), or 100 micrograms ALHRH + 50 micrograms EB; males-vehicle, 100 micrograms ALHRH, 500 micrograms testosterone propionate (TP), or 100 micrograms ALHRH + 500 micrograms TP. ALHRH blocked the selective increase in plasma follicle-stimulating hormone (FSH) observed in female rats as well as the parallel increases in both gonadotropins seen in male rats following castration. Administration of EB or ALHRH + EB to females significantly suppressed both gonadotropins compared with control levels. However, EB alone did not completely block the rise in plasma FSH in females. In males, all three treatments significantly suppressed the increases in both gonadotropins when compared with control levels. These data demonstrate that hypothalamic LHRH plays an essential role in the acute elevations of plasma gonadotropins following castration in rats. In addition, these data suggest that the selective rise of FSH in females is dependent on LHRH stimulation of pituitary gonadotropes.     

Effects of acyclic analogs of atrial natriuretic factor on proliferative processes of the epithelial tissue in white rats]

The effect of atrial natriuretic factor (ANF) synthetic linear truncated analogues AP-H-6-OH and AP-FOR-6-OH on corneal, skin, duodenum and colon epithelium proliferation has been studied on male rats. The epithelium mitotic activity and DNA synthesis were evaluated 4 and 24 h after intraperitoneal injection of 10 or 100 micrograms/kg peptides. In a dose of 10 micrograms/kg both ANF synthetic analogues inhibited proliferation processes in corneal epithelium, but activated the DNA synthesis in duodenum and colon epithelium. AP-FOR-6-OH (10 micrograms/kg) decreased the mitotic activity of skin epithelium and increased the silver grain density over the cell nuclei at the same time. 100 micrograms/kg ANF analogues stimulated cell mitogenesis in all organs studied. According to the data obtained ANF linear truncated analogues influence on epithelium proliferation is similar to effector of previously studied cyclic atriopeptin AP II.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu(2) and Gly(7) in this lead peptide, cyclo(CH(2)CO-Glu(1)-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC(50)=6.5 microM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of omega-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu(1) and Adi for Glu(4) in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency (19, IC(50)=23.3 microM) relative to G1TE. Moreover, the lipophilic chain in omega-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.     

Development of highly potent and selective dynorphin A analogues as new medicines.

Dynorphin A (Dyn A), a 17 amino acid peptide H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH, is a potent opioid peptide which interacts preferentially with kappa-opioid receptors. Research in the development of selective and potent opioid peptide ligands for the kappa-receptor is important in mediating analgesia. Several cyclic disulphide bridge-containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A(1-11)-NH2 cyclic analogues incorporating the sulphydryl-containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the mu- and delta-receptors) as the parent Dyn A(1-11)-NH2 peptide in the guinea-pig brain (GPB), but exhibited a much lower activity in the guinea-pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure-activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at kappa-opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand-based drug design.     

On the role of basic residues of head-to-tail cyclic bradykinin analogues on rat duodenum relaxation

Summary Three linear bradykinin (BK) analogues, Lys-Lys-BK, Nle-Lys-BK and Lys-Nle-BK and their head-to-tail cyclic analogues, along with cyclo-Nle-Nle-BK and cyclo-Lys-Lys-[Trp⁵]BK, were synthesized and tested on an isolated rat duodenum preparation. All kinins, except the [Trp⁵]-analogue, cause relaxation with EC₅₀ values in the picomolar range. The most potent linear analogue (Lys-Nle-BK) is about 40 times more active than BK and the most potent cyclic kinin (cyclo-Nle-Lys-BK) is about 6 times more active. Present results suggest that the significant potency of cyclo-Lys-Lys-BK, the earlier most potent cyclic kinin which is only a little less potent than linear BK, depends on the ring size rather than on the presence of the extra basic residues.     

Further characterization of the direct inhibitory effect of LHRH agonists at the testicular level in the rat

To further clarify the relative importance of the pituitary and gonadal sites of LHRH action, intact and hypophysectomized adult male rats were treated with hCG for 7 days, in the presence or absence of simultaneous treatment with increasing doses of the LHRH agonist [D-Ser(TBU)6des-Gly-NH2(10)]LHRH ethylamide, Buserelin (0.025, 0.25, 2.5 or 25 micrograms/rat, twice daily). Daily treatment of intact adult rats with hCG (25 IU) markedly increased ventral prostate and seminal vesicle weight, while a dose-dependent inhibition of the effect was observed following combined administration of Buserelin. In hypophysectomized rats, treatment with hCG resulted in a partial restoration of ventral prostate and seminal vesicle weight, while combined treatment with a high dose of the LHRH agonist (25 micrograms, twice daily) partially (P less than 0.05) inhibited the stimulatory effect of hCG. LH/hCG receptors were almost completely inhibited after hCG injection alone and a further decrease was observed in the presence of simultaneous LHRH agonist treatment. The hCG-induced stimulation of GH/PRL receptors was counteracted by Buserelin treatment in hypophysectomized animals. The present data demonstrate that although LHRH-induced LH release has been shown to play a major role in the loss of testicular functions induced by low doses of LHRH agonists in the rat, a direct inhibitory action of LHRH agonists can be exerted at the testicular level at high doses of the peptide.     

Effect of LHRH immunoneutralization on follicular development, the LH surge and luteal function in the stumptailed macaque monkey (Macaca arctoides)

A dose of 100 microliter of a potent ovine LHRH gamma globulin inhibited ovulation in the cyclic rat when administered at 12:00 h on the day of pro-oestrus. A dose of 10 ml of the preparation was administered i.v. to female stumptailed macaques to achieve circulating antibody titres 3-4-fold higher than in the rat. In an ovariectomized macaque, this caused a marked fall in serum concentrations of LH to less than 10% of pretreatment values and also a significant, though less pronounced, fall in FSH. Six monkeys were treated with the LHRH gamma globulin during the mid-late follicular phase of the cycle. In 2 monkeys in which serum oestradiol concentrations were less than 100 pg/ml at the time of antibody administration, the rising oestradiol levels were abruptly suppressed and the normal mid-cycle LH surge failed to occur. Serum concentrations of LH and FSH declined to low levels for 8-10 days after which time normal follicular development occurred. In the remaining 4 monkeys in which follicular development was more advanced as indicated by serum oestradiol concentrations of greater than 100 pg/ml, the antibodies induced either a transient decline or had no effect on the rising serum concentration of oestradiol. An LH/FSH surge followed by a rise in serum progesterone occurred in these macaques. When the antibodies were administered to a further 6 macaques, which had also been treated with oestradiol benzoate during the early follicular phase to induce an LH surge, the neutralization of LHRH again failed to block the surge even when the dose of antibody was increased to 20 ml. The results show that LHRH antibodies were unable to block the LH surge in the macaque. They contrast with results obtained with LHRH immunoneutralization in the sheep, rat, hamster, mouse and bird and suggest that the ability of oestrogen to induce an LH surge by acting directly on the LHRH-primed anterior pituitary gland is more dominant in the primate.     

Changes in prolactin levels caused by luteinizing hormone releasing hormone

The acute effects of luteinizing hormone releasing hormone (LHRH) on the release of prolactin (PRL) were investigated in 12 normal cycling women and 42 women with various menstrual disorders. LHRH (100 micrograms) was bolusly injected intramuscularly and PRL levels were measured immediately before the injection and at 30 minutes and 60 minutes after the injection. LHRH elicited an increase of more than 25% in PRL levels in 15 cases (27.8%) at both 30 minutes and 60 minutes after the injection, whereas PRL levels were decreased by more than 25% in 7 cases (13.0%). The PRL response to LHRH seemed to be related to basal PRL levels. Especially when the PRL concentration was 20 ng/ml or more, LHRH decreased PRL levels in 7 cases out of 16. On the other hand, LHRH increased PRL levels in the majority of cases with a PRL concentration less than 20 ng/ml. In conclusion, the LHRH injection occasionally alters PRL levels in either a positive or negative manner, depending upon the basal PRL levels.     

Consequences of increasing or decreasing plasma FSH concentrations during the preovulatory period in Romney ewes

Romney ewes were infused with ovine FSH (NIADDK-oFSH-16) for 48 h from the initiation of luteolysis with cloprostenol. Doses of 2.5 or 5 micrograms/h which partly or completely prevented the normal preovulatory decline in plasma FSH concentrations caused a significant increase in mean ovulation rates. Ovulation rates were not increased significantly if the FSH (5 micrograms/h) was infused for only 20 h starting from the initiation of luteolysis or 24 h later. Infusion of a less potent and relatively impure preparation of FSH (i.e. FSH-P) at 0.5 mg/h for 48 h after cloprostenol treatment also increased the mean ovulation rate significantly. However, if the FSH-P was given for only the first 24 h, or if the start of the infusion was delayed for more than 12 h, mean ovulation rates were not increased significantly. Infusion of LH (NIADDK-oLH-25, 5 micrograms/h) for 48 h from the initiation of luteolysis decreased the mean ovulation rate significantly. Administration of bovine follicular fluid to suppress plasma FSH concentrations below normal during the first 24 h after cloprostenol injection did not delay oestrus. However, oestrus was delayed by approximately 2 days if plasma FSH concentrations were reduced by bovine follicular fluid 24 h after the initiation of luteolysis. As ovulation rate increased, the mean weight of individual corpora lutea of each ewe decreased. In ewes with a single ovulation, most corpora lutea weighed greater than 600 mg, but as the ovulation rate increased the proportion of corpora lutea present weighing less than 400 mg rose steadily.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mouse epidermal growth factor on plasma concentrations of LH, FSH and testosterone in rams

Two experiments were conducted to examine the effects of mouse epidermal growth factor (EGF) on the concentrations of testosterone, LH and FSH in jugular blood plasma and on the pituitary responsiveness to LHRH. In 20 rams treated with subcutaneous doses of EGF at rates of 85, 98 or 113 micrograms/kg fleece-free body weight, mean plasma LH and testosterone concentrations were significantly reduced (P less than 0.05) at 6 h after treatment but not at 24 h. EGF treatment at 130 micrograms/kg fleece-free body weight suppressed the plasma content of these hormones for up to 48 h. Mean plasma FSH concentrations decreased significantly (P less than 0.05) for up to 48 h after EGF treatment, the effect being most pronounced in rams with mean pretreatment FSH values greater than or equal to 0.5 ng/ml. Intravenous injections of 1.0 micrograms LHRH given to each of 5 rams before and at 6 h, 24 h and 72 h after EGF treatment produced LH and testosterone release patterns which paralleled those obtained in 5 control rams similarly treated with LHRH. These results suggest that, in rams, depilatory doses of mouse EGF temporarily impair gonadotrophin and androgen secretion by inhibiting LHRH release from the hypothalamus. Such treatment appears to have no effect on the responsiveness of the pituitary to LHRH.     

Delay in the age of balano-preputial skinfold cleavage and alterations in serum profiles of testosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and gonadotropins in adult rats treated during puberty with luteinizing hormone releasing hormone

Previous work has shown that chronic treatment of intact, immature male rats with luteinizing hormone releasing hormone (LHRH) decreases sex accessory gland weights and results in retardation of the normal developmental increase in the ratio of serum testosterone (T)/5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Diol) via an apparent enhancement of testicular 5 alpha-reductase or 3 alpha-hydroxysteroid oxidoreductase activities. In the present work, androgen dependent balano-preputial skinfold cleavage was significantly delayed by approximately one week in intact, immature male rats which were treated daily for two weeks with either 1.0 micrograms, 2.5 micrograms or 5.0 micrograms of LHRH during a discrete phase of pubertal development (28-41 days of age). In intact, adult (62 day old) animals which received LHRH treatments during pubertal development, serum T concentrations and sex accessory gland weights were reduced compared to control animal values. Serum 3 alpha-Diol content in the adult rats was either unaltered or increased significantly depending on the LHRH dosage employed during sexual development. Serum luteinizing hormone concentrations were not different between control and LHRH-pretreated adult rats whereas the highest dosage of LHRH employed (5.0 micrograms) during puberty resulted in a significant elevation of adult serum follicle stimulating hormone levels. It is suggested that chronic LHRH treatment of the male rat during puberty results in a perturbation in testicular androgen biosynthetic activities and an impairment of pituitary-testicular hormone feedback mechanisms which persist at least through early adulthood.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.     

Design and synthesis of novel imidazoline derivatives with potent antihyperglycemic activity in a rat model of type 2 diabetes

Imidazoline derivatives have been reported to show antihyperglycemic activity in vivo. In the present study, we first showed that there was no correlation between the in vivo antidiabetic activity and the in vitro affinities for the I1/I2 binding sites for several substituted aryl imidazolines. Among these compounds, 2-(alpha-cyclohexyl-benzyl)-4,5-dihydro-1H-imidazole 2 exhibited potent antihyperglycemic properties. It was then chosen as lead compound. Thirty-six new derivatives were synthesized by replacing the cyclohexyl/benzyl group by various cyclic systems or the imidazoline ring by isosteric heterocycles. These compounds were evaluated in vivo for their antihyperglycemic activity using an oral glucose tolerance test (OGTT) in a rat model of type-2 diabetes obtained by giving a single intravenous (iv) injection of a low dose of streptozotocin to rats (STZ rats) and in normal rats. Nine compounds with an imidazoline moiety, possibly substituted by a methyl group, had a potent effect on the glucose tolerance in normal or STZ-diabetic rats, after an oral (po) administration of the test compound at a dose of 30 or 10 mg kg(-1), without any hypoglycemia. Replacement of the imidazoline ring by isosteric heterocycles resulted in a total loss of activity.     

Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells

The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells.     

Effects of 2(p-biphenyl) propionic acid on the life span of the corpus luteum of pseudopregnant and cyclic rats

A potent prostaglandin synthesis inhibitor (cyclooxygenase inhibitor), 2(p-biphenyl) propionic acid (BPPA), was administered subcutaneously at the dose of 1 mg twice daily to pseudopregnant and cyclic rats. It was found to prolong the duration of pseudopregnancy by about 10 days and to have little or no effect on estrous cycle length. It did not block ovulation and had little effect on ovulation rate in cyclic rats. BPPA was given to pseudopregnant rats in two trials (one in October-December and the other in March-May) to determine its effect on ovarian weight and plasma progesterone concentration on Days 14, 15 and 16 (Day 0=day of induction of pseudopregnancy). BPPA significantly (P less than 0.001) increased plasma progesterone concentration and reduced ovarian weight. The present data support the hypothesis that prostaglandins cause the normal functional demise of the corpora lutea of pseudopregnancy in the intact rat, and that depressing their synthesis will prolong the functional life span of the corpora lutea.