Role of GnRH in the regulation of pituitary GnRH receptors in female mice

The fall in pituitary GnRH receptors in female mice after ovariectomy (Ovx) was further decreased (greater than 50%), rather than prevented, by treatment with a GnRH antiserum, despite suppression of the post-gonadectomy increase in serum gonadotrophins, suggesting that increased endogenous GnRH secretion is not the mediator of GnRH receptor fall after ovariectomy in mice. Furthermore, GnRH antiserum reduced GnRH receptors by 30-50% in intact normal females, without altering receptor affinity, and rendered serum LH and FSH undetectable but did not reduce receptors in GnRH-deficient, hpg mice. When GnRH was administered to ovariectomized mice this failed to restore receptor values (fmol/pituitary) (intact = 55.3 +/- 2.4; Ovx = 30.1 +/- 2; Ovx + GnRH = 31.6 +/- 2.8), but serum LH was reduced from high post-ovariectomy values (231 +/- 42 ng/ml) to values normal for intact females (24 +/- 2 ng/ml). In contrast, multiple GnRH injections to intact female mice increased GnRH receptor by 35%, while serum LH was reduced to just detectable levels. A marked dissociation between GnRH receptor and serum gonadotrophin concentrations was observed. Administration of oestrogen (E2) plus progesterone (P) to ovariectomized mice in which endogenous GnRH had been immunoneutralized reversed the inhibitory effect of GnRH antiserum on GnRH receptors and increased values above those of ovariectomized controls, although no increase in serum or pituitary gonadotrophin levels was seen in ovariectomized mice treated with E2 + P + GnRH antiserum. Treatment with E2 and P of intact females receiving GnRH antiserum did not prevent the inhibitory effect of antiserum on receptors, while E2 + P treatment alone of intact female mice reduced GnRH receptors by 30%. These data suggest that the gonadal steroids reduce GnRH receptors in intact female mice by inhibiting hypothalamic GnRH secretion, and that a certain degree of pituitary exposure to GnRH is required for maintenance of a normal receptor complement. These results suggest that (1) the fall in GnRH receptors after ovariectomy is primarily attributable to removal of gonadal factors. The fall is not a reflection of alteration in endogenous GnRH interaction with the gonadotroph; (2) homologous ligand 'up-regulation' of GnRH receptors in female mice depends upon the presence of the ovaries; (3) endogenous GnRH is also required for GnRH receptor maintenance in intact female mice; and (4) GnRH receptor and serum gonadotrophin responses to hormonal changes can be dissociated and their relationship is complex.     

Molecular Recognition Theory of the complementary (antisense) peptide interactions

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203–207; Nature Med. 1 (1995) 876–878; Biochem. J. 234 (1986) 679–683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5′→3′ direction) and antisense peptides coded in 3′→5′ direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide–receptor systems with verified ligand–receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an “in vivo” experiment. It was shown that 3′→5′ peptide antisense of -MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.     

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT_1A receptor. Both antisera allowed the immunoprecipitation of 5-HT_1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT_1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT_1A sites labelled by selective radioligands such as [³H]8-OH-DPAT and [¹²⁵I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT_1A receptor binding subunits with G proteins from one brain area to another.     

Structure-function studies of linear and cyclized peptide antagonists of the GnRH receptor.

Structurally new analogs of the peptidic GnRH receptor antagonist Cetrorelix as well as conformationally constrained cyclized deca- or pentapeptides were synthesized and selected peptides evaluated comprehensively. To understand how structural variations of the antagonistic peptide effect pharmacodynamic properties, binding affinities and antagonistic potencies toward the human and rat GnRH receptor were determined. Whereas large substituents in position 6 of linear peptides are compatible with high binding affinity (K(D) < 0.5 nM), all cyclized peptides except the cyclo[3-10] analog D-52391 depicted low binding affinity (K(D) > 10 nM). Binding affinity and antagonistic potency in vitro correlated for all peptides and surprisingly no discrimination between human and rat receptor proteins was observed. Since receptor residues W(101) and N(102) are involved in agonist and antagonist binding, equally potent but structurally different antagonists were tested for binding to the respective W(101)A and N(102)A mutants. In contrast to linear decapeptides, residues N(102) and W(101) are not involved in binding of D-23938 and W(101) is the critical residue for D-52391 binding. We conclude that although equally potent, peptidic GnRH receptor antagonists do have distinct interactions within the ligand binding pocket. Finally, selected antagonists were tested for testosterone suppression in male rats. The duration of testosterone suppression below castration levels differed largely from 1 day for Ganirelix to 27 days for D-23487. Systemic availability became evident as the most important parameter for in vivo efficacy.     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

In vitro LH release and cAMP accumulation induced by synthetic GnRH derivatives

The LH-releasing activity of GnRH and nine synthetic GnRH derivatives was tested in pituitary monolayer cell culture prepared from female rats. D-amino acid-substituted analogs were found to be 12 to 18-fold as active as GnRH, while D-amino acid GnRH-[1-9]-ethylamide analogs showed 15 to 38-fold activity as compared to GnRH. Dehydroproline-GnRH was equipotent with the parent compound. Asp(A)6-GnRH-EA was less active than GnRH and it was a partial agonist only. All peptides stimulated intracellular cAMP content of the cultured cells at 1 hr and 4 hr of incubation. A nearly uniform 1.8 to 2-fold increase above basal cAMP could be observed with all peptides tested at their maximally active concentrations. However, no correlation could be established between the relative LH-releasing activities and cAMP-elevating potencies of the peptides. The findings suggest that cAMP may not be involved in overall LH-release by GnRH but leave the possibility open that cAMP could be involved in certain steps of mobilizing compartmentalized LH pools of pituitary gonadotrophs.     

An enzyme-linked immunosorbent assay for plant cadmium-binding peptide

Polyclonal antibodies raised against Cd-binding peptide from roots of Agrostis gigantea Roth were used with an enzyme-linked immunosorbent assay (ELISA). The antigen was best adsorbed to Immulon 2 “U” microtitre plates in 50 mM acetic acid. The antibodies to the antigen from Agrostis cross-reacted with Cd-binding peptides from the roots of maize and tomato, but not with glutathione nor metallothioneins I and II from rabbit liver. The antibodies reacted specifically with peptides rich in cysteine and glutamate, and having glycine or serine in the least amount. Reaction of antibodies was limited to ELISA; the antiserum did not form antigen-antibody precipitates when tested by standard diffusion and immunoelectrophoretic methods.     

Insect oostatic activity of GnRH and its fragments

Summary Mammal,¹²⁵I-mammal, salmon, chicken I and II GnRHs and three fragments of mammal GnRH were synthesized and their effect on oogenesis in the flesh flyNeobellieria (formerlySarcophaga) bullata (Diptera) was investigated. The peptides were prepared by the Merrifield solid phase synthesis on polystyrene/divinylbenzene polymer using the N^α-Boc strategy in DMF and were purified by preparative RP-HPLC in a gradient of water-MeOH. From the peptides assayed, only mammal GnRH and two of its carboxy-terminus truncated analogs remarkably affected the processes of egg development in ovarioles, causing changes in the follicular epithelium, proliferation of its nuclei and cell division towards the inner part of the egg chamber. The process led to the occurrence of multinuclear follicular epithelium which finally filled up almost the whole egg chamber and then it degenerated. The inability of GnRH of other animal species to evoke the changes in the egg development establishes the question of primary structures of GnRH responsible for these biological effects. The identity of sequences of GnRHs from position 1 up to 6 (with the exception of chicken GnRH II) points to functionality of amino acids located in positions 7 and 8 of the peptide chain. The radioactivity of the¹²⁵I-labelled mammal GnRH with maintained oostatic activity and its receptor competition with the non-labelled mammal GnRH were measured in slected insect organs and exhibited different residual values according to the organ and the time after application of the peptide. A transfer of the radioactivity into the next (F₁) generation was also observed. Abbreviations:AAA, amino acid analysis; ACN, acetonitrile; Boc, tert-butyloxycarbonyl; BrZ, 2-bromobenzyloxycarbonyl; Bzl, benzyl; CZE, capillary electrophoresis; DCC, N,N-dicyclohexylcarbodiimide; DCM, dichloromethane; DIEA, N-ethyl-diisopropylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; FAB MS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; For, formyl; FSH, folicule stimulating hormone; GnRH, gonadotropin-releasing hormone; HOBt, 1-hydroxybenzotriazole; LH, luteinizing hormone; RP HPLC, reversed phase high performance liquid chromatography; TFA, trifluoroacetic acid; Tos, 4-toluenesulfonyl. The symbols of amino acids and peptides are in accordance with the 1983 recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Eur. J. Biochem, 138 (1984) 9)     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

GnRH-prohormone-containing neurons in the primate brain: immunostaining for the GnRH-associated peptide

The structure of the prohormone for mammalian gonadotropin releasing hormone (proGnRH) includes the GnRH decapeptide followed by a 56 amino acid GnRH-associated peptide (GAP). In this study, we compared immunostaining of brain neurons and fibers for GAP and GnRH in fetal rhesus monkeys and juvenile baboons. We used antisera against different portions of human and rat GAP (proGnRH 14-24, proGnRH 40-53, and proGnRH 52-66) or against GnRH and the PAP technique. Liquid phase absorption with GAP or GnRH confirmed the specificity of these antisera. Major accumulations of GAP immunoreactive (GAP+) perikarya occurred in the medial septal and preoptic areas and the nucleus of the diagonal band of Broca (44.6% in rhesus, 49.6% in baboon), supraoptic region including the area dorsal to the optic tract (21.9% in rhesus, 23.0% in baboon), and the medial basal hypothalamus (15.7% in rhesus, 16.4% in baboon), especially at the infundibular lip. Occasional cell bodies were scattered throughout the hypothalamic and forebrain regions studied. GAP+ fibers were widely distributed, but formed well-defined pathways such as the periventricular and ventral hypothalamic tract. In addition, GAP+ nerve terminals with various densities occurred in the lamina terminalis, the zona externa of the infundibulum, and behind the infundibular stalk. Fetal rhesus macaques had more GAP+ cell bodies, denser fiber networks, and more distinct pathways than juvenile baboons. However, fiber and terminal immunostaining was somewhat less intense for GAP than GnRH in comparable regions. These results indicate that proGnRH (GAP) is present in the same population of neurons as GnRH in the primate brain. They also suggest that post-translational products of proGnRH are present in perikarya, axons and terminals, and that GnRH and GAP and/or further cleavage products are consecreted into hypophysial portal blood in the primate.     

C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor

We have previously described that [Tyr⁰]CGRP(28–37) acts as a receptor antagonist of rat CGRP in guinea pig pancreatic acini. We therefore examined other C-terminal peptides of CGRP for such activity. CGRP-acetyl(28–37) acetate did act as a rat CGRP antagonist. However, C-terminal CGRP peptides of 4 to 8 amino acid residues did not antagonize the actions of rat CGRP but stimulated amylase secretion. In pancreatic acini, a maximally effective concentration of rat CGRP (100 nM) caused a 2.1-fold increase in amylase secretion. When the C-terminal peptides of CGRP were tested in at 100 μM, CGRP(34–37) caused a 1.8-fold increase in amylase secretion, CGRP(33–37) a 2.8-fold increase, CGRP(32–37) a 9.2-fold increase, CGRP(31–37) a 4.1-fold increase, and CGRP(30–37) a 5.1-fold increase. Further studies with the most effective peptide, CGRP(32–37), demonstrated that it did not cause release of lactate dehydrogenase, and thus did not cause amylase release by cell damage. Unlike rat CGRP, CGRP(32–37) did not increase cellular cyclic AMP, but did stimulate outflux of ⁴⁵Ca. CGRP(32–37)-stimulated amylase release was not inhibited by the substance P receptor antagonist, spantide, by the bombesin receptor antagonist, [D-Phe⁶]bombesin(6–13) propylamide, or by the muscarinic receptor antagonist, atropine, but was inhibited by the CCK receptor antagonist L364,718. C-terminal peptides of CGRP inhibited binding of ¹²⁵I-BH-CCK-8, with the relative potencies of the peptides being the same as their relative potencies for stimulating amylase secretion. The present data demonstrate that C-terminal peptides of CGRP, although they have only 2 amino acid residues in common with CCK(26–33), act exclusively at CCK receptors on pancreatic acini to stimulate amylase secretion.     

Insect oostatic activity of GnRH and its fragments

Mammal, ¹²⁵I-mammal, salmon, chicken I and II GnRHs and three fragments of mammal GnRH were synthesized and their effect on oogenesis in the flesh fly Neobellieria(formerly Sarcophaga) bullata (Diptera) was investigated. The peptides were prepared by the Merrifield solid phase synthesis on polystyrene/divinylbenzene polymer using the N-Boc strategy in DMF and were purified by preparative RP-HPLC in a gradient of water-MeOH. From the peptides assayed, only mammal GnRH and two of its carboxy-terminus truncated analogs remarkably affected the processes of egg development in ovarioles, causing changes in the follicular epithelium, proliferation of its nuclei and cell division towards the inner part of the egg chamber. The process led to the occurrence of multinuclear follicular epithelium which finally filled up almost the whole egg chamber and then it degenerated. The inability of GnRH of other animal species to evoke the changes in the egg development establishes the question of primary structures of GnRH responsible for these biological effects. The identityof sequences of GnRHs from position 1 up to 6 (with the exception of chicken GnRH II) points to functionality of amino acids located in positions 7 and 8 of the peptide chain. The radioactivity of the ¹²⁵I-labelled mammal GnRH with maintained oostatic activity and its receptor competition with the non-labelled mammal GnRH were measured in selected insect organs and exhibited different residual values according to the organ and the time after applicationof the peptide. A transfer of the radioactivity into the next (F₁) generation was also observed.     

GnRH dose reduction decreases pituitary LH release and ovulatory response but does not affect corpus luteum (CL) development and function in llamas

Gonadotrophin releasing hormone (GnRH) is commonly used in llamas to induce ovulation; however, the consequence of reduced doses of GnRH on luteinizing hormone (LH) release, ovulatory response, and subsequent corpus luteum (CL) development and function have apparently not been investigated. Hence, we examined the effect of gradual reduction of gonadorelin acetate (GnRH) dosage on pituitary LH release, ovulatory response, CL development, and plasma progesterone concentrations in llamas. Non-pregnant, non-lactating adult llamas were examined once daily by transrectal ultrasonography, and those with a follicle ≥8 mm in diameter that had grown for three consecutive days were randomly assigned to receive 50 (GnRH50, n = 23), 25 (GnRH25, n = 29), 12.5 (GnRH12.5, n = 29), or 6.25 μg (GnRH6.25, n = 29) of GnRH, or 0.5 mL of PBS (Control group, n = 16) im. In a subset (7 or 8 animals/group), intense blood sampling was done to measure LH concentrations. All females were examined by ultrasonography every 12 h from treatment (Day 0) to Day 2 to determinate ovulation, and thereafter on alternate days until Day 16 to evaluate CL development (9-13 animals/group). Also, blood samples for progesterone determination were taken (9 or 10 animals/group) on alternate days from Days 0-16. Ovulatory response (%) was highest (P < 0.05) in the GnRH50 (82.6), intermediate in the GnRH25 (72.3) and GnRH12.5 (75.9) groups, and lowest in the GnRH6.25 group (48.3). No ovulations were detected in the Control group. Mean peak LH concentrations (ng/mL) were highest (P < 0.05) for GnRH50 (6.2), intermediate for GnRH25 (4.4) and GnRH12.5 (2.9), and lowest for GnRH6.25 (2.2) groups. In addition, based on regression analysis, llamas with an LH peak <4 ng/mL were less likely to ovulate. Llamas given 50 μg of GnRH released more (P < 0.05) pituitary LH and had an LH surge of longer duration than those given 25, 12.5, or 6.25 μg. However, in those that ovulated, neither GnRH treatment nor treatment by time interaction affected (P > 0.05) CL diameter or plasma progesterone concentrations. In summary, reducing the dose of GnRH gradually decreased the magnitude of the preovulatory LH surge and ovulatory response; however, subsequent CL development and plasma progesterone concentrations were not affected.     

D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea

Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.     

Physiological role of putative testicular gonadotrophin releasing hormone (GnRH)

Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non-existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular effects of pituitary adenylate cyclase activating peptide: a comparison with vasoactive intestinal peptide

The effects of pituitary adenylate cyclase activating peptide (PACAP) on the blood pressure of the anesthetized rat and on the isolated rat tail artery were investigated and compared to those of vasoactive intestinal peptide (VIP). PACAP-38, PACAP-27 and the C-terminal fragment 16–38 caused a dose-dependent decrease in the systemic blood pressure. PACAP-27 and PACAP-38 were equipotent with VIP. The C-terminal fragment 16–38 was much less potent than VIP. The duration of action was longer for equimolar doses of PACAP-38 and PACAP-27 than for VIP and much longer than for PACAP 16–38. PACAP-27 and the phosphodiesterase inhibitor rolipram given in combination produced additive vasodepressive responses. In vitro PACAP-38, PACAP-27, VIP and PACAP 16–38 relaxed the phenylephrine-precontracted rat tail artery. PACAP-38 and PACAP-27 were equipotent with VIP. PACAP 16–38 was much less potent than the full-length peptides. The responses were resistant to atropine and propranolol. Addition of VIP 1 μM to preparations exposed to 1 μM PACAP-38 or -27 did not produce a further relaxation. VIP-like peptides, PACAP in particular, are known to activate adenylate cyclase and to elevate the plasma cyclic AMP (cAMP) concentration. cAMP was found to be a potent vasodepressor in the anaesthetized rat and a potent vasodilator of precontracted blood vessels. On the basis of these results it cannot be excluded that the vascular effects of PACAP are secondary to the effect of elevated levels of extracellular cAMP.     

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.     

Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.