Two isoforms of gonadotropin-releasing hormone are coexpressed in neuronal cell lines.

GnRH-I serves as the neuropeptide that regulates mammalian reproduction. Recently, several groups have identified in the brain of rodents, monkeys, and humans a second isoform of GnRH (GnRH-II) whose structure is 70% identical to that of GnRH-I. In this study we demonstrate for the first time human and mouse neuronal cell lines that express both GnRH-I and GnRH-II. Following the screening of several human neuronal cell lines by RT-PCR and Southern hybridization, we demonstrated that two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, coexpress messenger RNA encoding the two isoforms of GnRH. Nucleotide sequencing indicated that the complementary DNA fragments are identical to those of the known human GnRH-I and GnRH-II sequences. Extracts obtained from the TE-671 and LAN-1 cell lines as well as from the immortalized mouse hypothalamic GT1-7 neuronal cell line were found to contain the two isoforms of GnRH, which exhibited identical chromatographic properties as synthetic GnRH-I and GnRH-II, in HPLC followed by specific RIAs. Furthermore, double immunofluorescence studies demonstrated the two GnRH isoforms in LAN-1, TE-671, and GT1-7 cells. The identification of neuronal cell lines expressing both GnRH-I and GnRH-II provides tools for studying the differential regulation of gene expression and secretion and for studying the interaction between the two isoforms. Such studies may contribute to elucidation of the physiological functions of GnRH-II, which are still unknown.

Structure-activity studies of reduced-size gonadotropin-releasing hormone agonists derived from the sequence of an endothelin antagonist.

We have previously determined that Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide I), an endothelin antagonist, binds specifically (Ki = 1.9 microM) to the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Moreover, peptide I exhibits a GnRH agonistic activity, mediated directly by the GnRH receptor. We now report structure-activity studies of peptide I in respect to its interactions with the GnRH receptor. Our studies suggest that the bioactive conformation of peptide I, recognized by the GnRH receptor, is of a cyclic nature. Thus cyclic analogues of peptide I exhibit higher affinity to the GnRH receptor and increased agonistic potencies as compared to peptide I itself. A linear peptide, Ile-Ile-Trp-D-Trp-Leu-Asp, which presumably forms a similar cyclic conformation, was also shown to be a GnRH agonist. Intraperitoneal administration of Ac-Ile-Ile-Trp-D-Trp-Leu-Cys-OH (Ki = 0.32 microM), one of the cyclic hexapeptides that we have synthesized, to rats induces secretion of luteinizing hormone (LH) with a potency which is only 1 order of magnitude less than that of GnRH itself. Moreover, plasma levels of LH remained elevated for a longer period of time following the administration of the cyclic hexapeptide. This novel class of GnRH agonists may prove useful in the development of new therapeutics.

Design and synthesis of potent hexapeptide and heptapeptide gonadotropin-releasing hormone antagonists by truncation of a decapeptide analogue sequence.

A novel strategy for designing reduced-size analogues of the decapeptide gonadotropin-releasing hormone (GnRH) was developed. As opposed to previous attempts to delete residues from either of the peptide's termini, our approach is based upon the known importance of both C- and N-terminals of GnRH analogues for receptor recognition, whereas the central part of the molecule is replaced by a short spacer. The present truncation strategy was successful for generation of reduced-size hexapeptide and heptapeptide antagonists possessing potent antagonistic capacity. The same methodology was not suitable for the generation of reduced-size agonists, suggesting different conformational characteristics for GnRH agonists and antagonists. A heptapeptide antagonist designed by this method was shown to inhibit serum levels of luteinizing hormone in castrated rats in vivo. Structure-activity studies suggested that the structural preferences for GnRH receptor recognition are similar to those reported for decapeptide antagonists. Our studies resulted in a heptapeptide GnRH antagonist (Ac-D-Nal2-D-Cpa-D-Pal-Gly-Arg-Pro-D-Ala-NH2) with high receptor binding affinity (IC50 = 7 nM), as compared to that of GnRH itself (IC50 = 2 nM). The highest affinity of a hexapeptide antagonist that we have synthesized was somewhat lower (IC50 = 45 nM).

The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform.

The mammalian gonadotropin-releasing hormone (GnRH-I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH-III, in the brain of mammals is reported herein. GnRH-III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH-III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone-releasing activity of purified GnRH-III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH-III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH-I. Immunohistochemical studies demonstrated GnRH-III-containing neurons in the hypothalamus and midbrain of human and GnRH-III fibers in the median eminence of rats. The distribution of GnRH-III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic-pituitary axis, GnRH-III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.

Synthesis and bioactivity of fatty acid-conjugated GnRH derivatives.

Transdermal delivery of peptidic drugs is usually inefficient, notably due to their hydrophilic character which makes it difficult to cross the hydrophobic layers of the skin. In order to obtain transdermally deliverable analogs of gonadotropin releasing hormone (GnRH), we have synthesized related hydrophobic derivatives by attaching various aliphatic acids to the N(epsilon)-amino side chain of [D-Lys]6GnRH, a superactive GnRH agonist. It was found that the affinity toward the GnRH receptor gradually decrease with increased hydrophobicity, i.e. increase in chain length of the attached aliphatic acid. Nevertheless, analogs with 12-carbon or shorter aliphatic acids were shown to be GnRH superagonists, with in vitro and in vivo potencies similar to that of [D-Lys]6GnRH. [D-Lys-lauryl]6GnRH was shown to have a longer duration of action in vivo, as compared to [D-Lys]6GnRH. The transdermal penetration of the peptides was evaluated by in vivo functional experiments in rats. According to these studies the efficiency of penetration is gradually lowered in increasingly hydrophobic analogs. These results are discussed with respect to the circular dichroism spectra of the peptides in trifluoroethanol. The spectra of the aliphatic acid-conjugated superagonists examined do not express a significant tendency towards a beta-turn conformation, typical of GnRH and its agonists. This finding contradict previous publications which suggested a correlation between the conformations of GnRH analogs in trifluoroethanol and their biological activities.

A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents.

Gonadotropin-releasing hormone-I (GnRH-I), present in the mammalian hypothalamus, regulates reproduction. In this study we demonstrate, for the first time, that an additional isoform of GnRH, [His5, Trp7, Tyr8] GnRH-I (GnRH-II) is present in the brain of the mouse, rat and human. Human and rat brain extracts contain two isoforms of GnRH, GnRH-I and GnRH-II, which exhibited identical chromatographic properties to the respective synthetic peptides, in high performance liquid chromatography. Using immunohistochemical techniques we have found that GnRH-II is present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain. It is of interest to note that in the hypogonadal mouse, although the GnRH-I gene is deleted, GnRH-II is present. Substantial concentrations of GnRH-II are also present in the hypothalamus and stored in the human pituitary stalk or in the mouse median eminence. By using reverse transcription (RT)-PCR we have also found that while GnRH-II is not expressed in the cerebellum, it is expressed in all three structures of the brain stem: midbrain, pons and medulla oblongata.