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MicroRNAs (miRNA) are believed to play a crucial role in the cause and treatment of temporal lobe epilepsy (TLE) by controlling gene expression in different stages of the disease. To investigate role of miRNA in the latent stage following status epilepticus, we first compared microRNA expression profiles in mice hippocampus at 1 week after pilocarpine-induced status epilepticus (SE) vs. controls in hippocampal tissues using Exiqon miRCURY LNA™ miRNAs Array. Then, the target genes of altered miRNAs were predicted using both TargetScan 7.1 and miRDB V5, and were further selected by intersecting with another independent mRNA expression profile dataset from the samples at the same time point. We found out 14 common genes as down miRNA target (up-mRNA) and 4 common genes as up miRNA target (down mRNA) in SE mice. miR-669m-3p-TRHR (thyrotropin releasing hormone receptor), miR-669m-3p-B3galt2 (β-1,3-Galactosyltransferase 2), miR-105-PDPN (Podoplanin) and miR-883b-3p-CLEC-2 (C-type-lectin-like-2) were found to be potential molecular mechanisms to modulate the calcium signaling pathway, glycosylation pathways and chemokine mediated inflammatory processes in mice hippocampus at 1 week after pilocarpine-induced SE, respectively. Our results offered potential novel insights into the cellular events in the mice hippocampus mediated by miRNASs-target genes that shape SE-evoked epileptogenesis.

Thyroid hormone exerts a pleiotropic effect on development and homeostasis. A novel thyroid hormone receptor beta isoform (hereafter referred to as TR beta 4) was cloned using PCR from a human pituitary cDNA library as a template. Analysis of the PCR products revealed a 137-bp insertion, which contains a stop codon in the middle, between the 5th and 6th exons that encode the ligand-binding domain of TR beta. The corresponding sequence of this insertion exists within the 5th intron of the human TR beta gene and consensus splice sequences were found at the junction sites. RACE analysis revealed that TR beta 4 is a carboxyl-terminal splicing variant of TR beta 1. RT-PCR and Northern blot analyses indicate that TR beta 4 mRNA is expressed in various human tissues, and especially abundant in testis and skeletal muscle. The TR beta 4 protein was unable to bind thyroid hormone (T3) and transient transfection assays demonstrate that TR beta 4 construct does not mediate T3-dependent gene regulation. TR beta 4 weakly but significantly inhibited transcription mediated by functional TR. Thus, this novel isoform may modulate hormone action as an endogenous antagonist in the tissue or cellular context.

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that stimulates gonadotropic hormone secretion upon interaction with its membrane receptors (type I) on pituitary cells, thereby governing reproductive processes. A second releasing hormone (GnRH II) expressed in mammals was shown earlier to be expressed in nonmammals and to have its own receptor. Here we demonstrate that a second receptor (type II) gene is present in the human genome, and report the cloning and characterization of its cDNA from monkeys. The cDNA encodes a G-protein-coupled/7 transmembrane receptor having a C-terminal cytoplasmic tail; it resembles more closely the type II receptors of amphibians and fish (approximately 55% identity) than it does the type I receptor of humans (approximately 39%). The GnRH type II receptor proved to be experimentally functional with, and specific for, GnRH II. GnRH receptor type II RNA is expressed ubiquitously in human tissues. This is the first report of a GnRH type II receptor in mammals. Its identification will permit exploration of its role in regulating gonadotropin secretion, female sexual behavior, and tumor cell growth.

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.

Thyrotropin, by binding its specific receptor on the plasma membrane of the thyrocyte, regulates thyroid function and differentiation. In FRTL5 cells, thyrotropin down-regulates the thyrotropin receptor (TSHr) promoter activity and induces the transcription of the alpha form of thyroid hormone receptor (TR-alpha 1). In this study we show that the thyrotropin receptor down-regulation, induced by thyrotropin, is mediated by TR-alpha 1. The thyroid hormone receptor binds, in vitro, the thyrotropin receptor minimal promoter and inhibits promoter activity in cotransfections experiments in CV 1 cells. The inhibition is achieved only in the presence of the thyroid hormone. The TSHr promoter mutated in the thyroid hormone receptor binding site does not bind TR, in vitro, and its activity is not inhibited, in cotransfection experiments, in CV 1 cells. The same mutation abolishes the TSH mediated down-regulation of the TSHr promoter activity in FRTL5 thyroid cells. These results support the hypothesis of a regulatory short loop of thyroid hormone in thyroid cells.

We cloned a human thyrotropin-releasing hormone receptor (TRH-R) gene and its pituitary cDNA, and examined whether TRH-R mRNA may be expressed and function in the GH-secreting pituitary adenomas of patients with acromegaly. The human TRH-R consists of 398 amino acids and is a member of the G protein-coupled receptor family. The gene has a single intron in the coding sequence. An addition of TRH caused an obvious increase in intracellular calcium concentration in chinese hamster ovary cells expressing this receptor. Although highly homologous to the rat and mouse TRH-Rs, the human TRH-R has several differences in the carboxyl-terminal structure. The sequence and functioning analyses showed the same expression of TRH-R mRNA in the pituitary adenomas as that in the normal pituitaries, indicating that 1) some adenomas of patients with acromegaly express an intact and functional TRH-R, and that 2) the paradoxical response of GH secretion to TRH found in the patients might be a direct effect of TRH on TRH-R expressed in the adenoma.

Summary Thyroid hormone receptors are nuclear proteins which regulate transcription in a hormone dependent manner. The baculovirus expression system was used for the overexpression of the β1 isoform of the human thyroid hormone receptor. The baculovirus produced receptor binds tri-iodothyronine with high affinity, is specifically immunoprecipitated with a β1 specific antibody, and binds to DNA that contains a known thyroid hormone receptor recognition site. Large scale production and purification of baculovirus produced receptor will be useful for structure-function analyses and studies of transcriptional regulation.

We have previously reported a family, Kindred A, with autosomal dominant generalized thyroid hormone resistance in which affected members were found to have a mutation in the carboxy-terminal domain of the c-erbA beta thyroid hormone receptor. In the current study, the thyroid hormone and DNA-binding properties of this mutant receptor were determined using c-erbA beta protein synthesized in vitro. Both the wild-type human placental c-erbA beta and Kindred A receptors bound [125I]-triiodothyronine, although the Kindred A receptor had decreased affinity for the hormone. The affinity for triiodothyronine was 4.5 x 10(9) M-1 and 2.3 x 10(10) M-1 for the mutant and wild-type receptors, respectively. No abnormality of DNA-binding was detected with the Kindred A receptor using a sensitive avidin-biotin DNA-binding assay with DNA fragments containing thyroid hormone response elements. The Kindred A mutant receptor which displays abnormal triiodothyronine-binding but normal DNA-binding activities in vitro acts as a dominant negative inhibitor of thyroid hormone action in man.

Although crucial for TH homeostasis, the molecular mechanisms responsible of thyroid hormone receptors (TRs)-mediated regulation of the hypothalamic – pituitary – thyroid axis (HPT) axis remain unclear. We examined the role played by TR-isoforms in combination with RanBPM, a novel coactivator of TRs. In transient transfections studies with the human TRH and TSH-α subunit promoters, we found that the overexpression of RanBPM increases the transcriptional activity of all TR-isoforms by a magnitude of 1.7- to 3-fold. The addition of RanBPM, in the absence of THs, increased the ligand-independent activation (LIA) of TRα1 and TRβ1 on both promoters tested by 300% and 200%, respectively, whereas, the LIA of TRβ2 was not significantly modified. This data reinforces the concept of isoform-specific regulation of genes of the HPT axis and demonstrates that RanBPM may be an important factor to achieve adequate regulation of nTREs in the presence of low TH levels.

Luteinizing hormone and gonadotropin releasing hormone receptors (LHR and GnRHR, respectively) are G protein-coupled receptors with important functions in reproduction. We have developed chimeric GnRHR-LHR that contain the full GnRHR coupled to various forms of the LH receptor C-terminus to explore the role of the LH receptor C-terminus in raft localization of the receptor and signaling. Addition of the full-length LHR C-terminus to GnRHR resulted in localization of the resting chimeric receptor in the bulk membrane rather than plasma membrane rafts as has been reported for the wild-type GnRHR [A. Navratil, S. Bliss, K. Berghorn, J. Haughian, T. Farmerie, J. Graham, C. Clay, M. Roberson, Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK, J. Biol. Chem. 278 (2003) 31593-31602]. With truncation of the LHR C-terminus, approximately 3% of chimeric receptors appeared in low density membrane fractions. Palmitoylation of sites on the LHR C-terminus appears important for raft localization. Mutations to C-terminus palmitoylation sites eliminated translocation of LH receptors from the bulk membrane to rafts upon binding of hCG although these mutant receptors retained the ability to signal via cAMP.

Parathyroid hormone (PTH) has a central role in the regulation of serum calcium and phosphate, while parathyroid hormone-related peptide (PTHrP) has important developmental roles. Both peptides signal through the same receptor, the PTH/PTHrP receptor (a class B G-protein-coupled receptor). The different biological effects of these ligands result from their modes of regulation and secretion, endocrine vs. paracrine/autocrine. The importance of PTH and PTHrP is evident by the variety of clinical syndromes caused by deficiency or excess production of either peptide, and the demonstration that intermittent injection of PTH increases bone mass, and thus provides a means to treat osteoporosis. This, in turn, has triggered increased interest in understanding the mechanisms of PTH/PTHrP receptor action and the search for smaller peptide or non-peptide agonists that have efficacy at this receptor when administered non-parenterally.

A new subfamily member of thyrotropin releasing hormone (TRH) receptor gene,TRHR2, was isolated from rat brain cDNAs. The deduced amino acid sequence ofTRHR2is 51 % identical to that of rat TRH receptor gene which was reported previously. Northern blot analysis withTRHR2probe revealed brain-specific expression of a 9.5 kb mRNA. In a binding experiment using the TRHR2-expressing COS cells, specific binding of TRH to TRHR2 was observed with Kd value of 9 nM which was equivalent to the Kd value (= 13 nM) of TRH binding to the TRH receptor previously reported. The active metabolite of TRH, histidyl-proline diketopiperazine, or cyclo(His-Pro), showed no specific binding activity. These results suggest that TRHR2 is a novel subtype of TRH receptor.

Unlike the wild-type thyrotropin (TSH) receptor, the chimeric TSH-LH/CG receptor TSH-LHR-14 does not cleave into two subunits when cross-linked to [125I]TSH on the surface of intact cells. Immunoblotting of TSH-LHR-14 in whole cell homogenates demonstrated that only a single chain receptor was detected under reducing conditions. TSH-LHR-14, like the A subunit of another chimeric receptor (TSH-LHR-10) that does cleave into two subunits, was almost entirely resistant to endoglycosidase H, indicating that it contains predominantly complex carbohydrate. The fact that TSH-LHR-14 reaches the cell surface where it binds TSH with high affinity and transduces a signal indicates that receptor cleavage into two subunits is not a prerequisite for TSH action.

Mutations involving the transmembrane domain of the thyrotropin receptor (TSHR) confer constitutive activation of the receptor and can cause human diseases. Naturally occurring activating mutations identified to date are located only in the transmembrane domain of the receptor. We now report a mutant involving the extracellular domain of the TSHR which also shows constitutive activation. This mutation is missing residues 339-367 located in the C-terminal portion of the extracellular domain. When expressed in COS-7 cells, the mutated TSHR (M3B) retained similar TSH binding ability to that of the wild-type receptor. However, the basal cAMP production without TSH stimulation in COS-7 cells transfected with M3B cDNA was significantly higher than that of COS-7 cells with wild-type receptor, indicating that the mutant receptor is constitutively activated. Our results provide new insight into the mechanism of receptor activation.

A cDNA encoding the human thyrotropin-releasing hormone receptor (hTRH-R) was isolated from a human brain cDNA library. Screening of 1.2 million clones resulted in 2 candidates. The largest clone contained TRH-R homologous sequences starting in the third transmembrane domain and included a long 3′ untranslated sequence. The smaller clone contained a potential start of the open reading frame, but was interrupted by an intron in the sixth transmembrane domain. The two clones had 497 bp of overlapping identical sequences and it was possible to assemble a complete cDNA thus restoring the assumed coding sequence. Electrophysiological studies of frog oocytes injected with in vitro transcribed mRNA showed TRH-specific inward currents, demonstrating that the reconstituted cDNA encoded a functional receptor. The predicted amino acid sequence of the hTRH-R protein showed high homology with the rat and mouse TRH-Rs with the exception of their C-terminal region. The human TRH-R gene seems to contain two introns.

The sequence from -1 to +6 bp in the hTSH beta gene contains overlapping putative thyroid hormone and AP-1 response elements. We demonstrate interaction between the AP-1 constituents c-fos and c-jun and thyroid hormone receptor in this region by transient transfection experiments using a -125 to +37 bp hTSH beta fragment. T3 inhibition was completely abolished by c-jun, but increased threefold by c-fos. A single transversion mutation at +2 bp restored T3 inhibition in the presence of c-jun and markedly reduced binding of purified c-jun by gel mobility shift assay. Thus, c-fos and c-jun influence T3 inhibition of hTSH beta expression in opposite directions acting through a response element shared with thyroid hormone receptor. Control of the relative cellular levels of these two proto-oncogenes may play a major role in modulating thyroid hormone inhibitory responses.

Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7-transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications.

We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10 −9 and 10 −7 M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation ofTTRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.

In vitro transcription/translation, using rat thyrotropin receptor cDNA, results in the formation of nonglycosylated proteins able to bind thyrotropin, one of which approximates the 87 Kd size predicted for the receptor. In the presence of canine pancreatic microsomal membranes, putative glycosylation sites are modified as evidenced by digestion with endoglycosidase H. Using a deletion mutant, the presence of a hydrophobic peptide after the initiation signal is established as a signal peptide critical to post translational processing by the canine pancreatic membranes but not to binding thyrotropin.

Three analogs (cpc-His-pyr, cpc-His(τMe)-pyr, cpc-Thi-pyr) of the thyrotropin releasing hormone, TRH (pGlu-His-Pro-NH2), have been synthesized and studied for TRH antagonistic activity in monkeys and in vitro, using isolated rat pituitaries. cpc-Thi-pyr inhibits the TRH induced release of TSH in vitro, cpc-His-pyr has inhibitory activity only in some experiments, while cpc-His(τMe)-pyr has none. Both cpc-Thi-pyr and cpc-His(τMe)-pyr appear to have slight agonistic activity. The TRH induced release of prolactin in monkeys could not be inhibited at dosages 1000-fold that of TRH.

Homogenates of human luteal tissue bound radioiodinated luteinizing hormone releasing hormone agonist. Specific binding was both time- and temperature- dependent. Native LHRH and two LHRH agonists competed for binding, whereas TRH, somatostatin and oxytocin did not, indicating that the binding sites were specific. The apparent Ka values were 2 × 107M−1 for both LHRH agonists and 106M−1 for native LHRH. This is the first demonstration of specific binding of LHRH to human ovarian tissue. No binding could be detected to ovarian tissue from postmenopausal women.

Receptors for thyroid hormones were extracted by 0.4 M KCl from the nuclei of rat liver and brain, and their binding properties compared to the properties of these receptors in unextracted nuclear suspensions. The inhibititory effect of a non-iodinated thyroid hormone analogue, 3,5,dimethyl-3′-isopropyl-l-thyronine (DIMIT) on [ 125 I]-T 3 binding was observed in the nuclear suspension of brain, but absent when the solubilized receptors of the same organ were tested. The initial properties of the receptor could be restored in a system containing the receptor and the extracted chromatin. Moreover, when the liver solubilized receptor was supplemented with the brain chromatin extract, the hepatic receptor acquired the binding ability of the brain receptors. The data suggest that chromatin associated components may confer organ specificity in thyroid hormone effects, and play a role in the selectivity of the recognition of thyroid hormone analogues by the receptor.

12-0-Tetradecanoylphorbol 13-acetate and thyrotropin releasing hormone were compared as thyrotropin secretagogues in cultured rat pituitary cells. The maximal secretion evoked by the former was only half that evoked by the latter. A23187 plus 12-0-tetradecanoylphorbol 13-acetate together produced as large a response as thyrotropin releasing hormone. Triiodothyronine inhibited phorbol ester-induced thyrotropin secretion, and this inhibition required protein synthesis.

Mouse uterus estradiol receptor undergoes a inactivation-reactivation process “in vitro”. The specific estrogen binding activity inactivated by nuclei, apparently through a dephosphorylation process (1,2,3), is reactivated by an ATP-dependent process. The enzyme reactivating the receptor has been purified from calf uterus cytosol. It shows high affinity for the inactive receptor (K m of ∼ 0.3 × 10 −9 mol of 17β-estradiol binding sites/l); it is simulated by MgCl 2 and CaCl 2 . Present and previous results suggest that in cytoplasm of intact cells a phosphorylation process makes the receptor able to bind hormone and in nuclei dephosphorylation of receptor causes loss of hormone binding activity.

The solubilized (“cytosolic”) receptor present in the rabbit uterus in the absence of hormone and the chromatin-bound (“nuclear”) receptor obtained after injection of a progestin were compared. Crude cellular extracts were analyzed by immunoblotting and receptors were purified by immunoaffinity chromatography. With both methods it was observed that the electrophoretic mobility of the “nuclear” receptor was slower than that of the “cytosolic” receptor. This difference in mobility appeared to be due to the existence of variably phosphorylated forms of receptor. The phosphorylation reaction was examined in uterine slices. In the absence of hormone the cytosolic receptor was phosphorylated. When hormone was added the phosphorylation of receptor was markedly enhanced and the electrophoretic mobility of the “nuclear” receptor was decreased. These experiments thus show that the receptor in its “cytosolic” form is a phosphoprotein. Under the effect of the hormone the receptor is further phosphorylated on some supplementary site(s). This polyphosphoprotein is the chromatin-bound, putatively active, form of the receptor. In this respect the intracellular progesterone receptor is similar to various membrane receptors for hormones and growth factors which are phosphorylated upon binding of their ligand.

Ever increasing potency is an international goal for antagonists of the luteinizing hormone releasing hormone (LHRH). [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3,Ser4,Arg5,D-3-Pal6,Leu7,Arg8,Pro9,D-Ala10]-NH2 caused 60%/125 ng inhibition of ovulation in the rat, and appears to be the most potent antagonist yet described. Strategy of design was the replacement of D-Arg6 with D-3-Pal6 and of Tyr5 with Arg5. replacing Arg5 with His5 reduced activity by 50% at 250 ng. Both the Arg5 and His5 analogs showed 100% inihibition of ovulation at 0.5 μg. Of ten pairs of analogs with D-3-Pal6 and D-Arg6, 3 10 with D-3-Pal6 were more potent than those with D-Arg6. Histamine release was less for the D-3-Pal6 peptides of three pairs.

Summary Gangliosides inhibit the binding of 125 I-labeled luteinizing hormone to rat testis membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with ganglioside can be detected by fluorescence spectroscopy. In both the binding inhibition and fluorescence studies, luteinizing hormone recognizes an oligosaccharide sequence on the ganglioside molecule distinct from that of thyrotropin, human chorionic gonadotropin, and cholera toxin.

Porcine hypothalamic fragments were extracted by 2M AcOH at 4°C, and the extractives were subsequently processed in the presence of one protease inhibitor and one anti-oxidant. Gel filtration was performed on Bio-Gel P-2, and supplementary [3H]-LHRH and [14C]

Summary The degradation of Luteinizing Hormone — Releasing Hormone (LH-RH) and several analogs by the adenohypophyseal pyroglutamate aminopeptidase, “LH-RH degrading endopeptidase” and post-proline-cleaving enzyme has been investigated using a fluorescamine-based assay. The rates of hydrolysis of these peptides have been compared, and the influence of amino acid substitutions or deletions in LH-RH on the enzymatic reactions is discussed. Superactive agonists modified in position 6 and 10 are degraded more slowly, but there is no strict colinearity between the resistance to degradation and the prolonged and enhanced biological activity of these analogs.

Leydig cells isolated from adult rat testes bound 125I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (KA=1.2 × 109M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with 125I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10−4M) concentrations but was inactive at 10−6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of 125I-LHRH agonist to Leydig cells in vitro by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.

A series of peptide analogs of luteinizing hormone releasing hormone (LH-RH), altered at position 6 and 10, was synthesized and evaluated in vivo for the ability to induce ovulation in the diestrous rat and in vitro for ability to release pituitary luteinizing hormone and follicle stimulating hormone. All the analogs with D-amino acid substitutions at position 6, even those with large bulky side chain, exhibited an amazingly high potency compared with the parent hormone, LH-RH. On the basis of the biological activities, structure-activity relationships in the central part of this molecule were discussed in detail.

Data show that the luteinizing hormone releasing hormone (LHRH) is biosynthesized by mitochondrial preparations from hypothalami which were obtained by centrifugation of a Ficoll gradient system. Incubation, extraction, use of carrier synthetic LHRH, and chromatography, yielded fractions showing associated hormonal activity and radioactivity. Other investigators reported biosynthesis of the thyrotropin and the growth hormone releasing hormones by soluble enzymes, but these enzymes might have been derived from mitochondria and/or synaptosomes. These particles appear implicated, but the true site of biosynthesis of these hormones is unknown. The mechanism at the site could possibly involve a pantetheine-protein template.

The synthesis of Val-His-Leu-Ser-Ala-Glu-Glu-Lys-Glu-Ala, isolated by Schally, et al. from porcine hypothalamus and reported by him to possess growth hormone releasing activity, is described. Similarities between this decapeptide and the amino-terminal sequence of the β-chain of porcine hemoglobin are pointed out. The syntheses of two analogs including the amino-terminal decapeptide of the β-chain of human hemoglobin are also described.

In order to localize and identify receptor structures, the binding of radiolabeled thyrotropin releasing hormone (TRH) to thyrotropin (TSH)-secreting cells from rat and bovine anterior pituitaries and from a mouse TSH-secreting tumor was studied in vitro . The binding of TRH to rat anterior pituitaries increased linearly with the log of TRH concentration in the incubation medium. Plasma membranes were the only subcellular fractions isolated after incubation from bovine anterior pituitary and the TSH tumor which bound detectable quantities of TRH.

The new L-glutaminyl-L-histidyl-L-prolinamide (Gln-His-Pro(NH 2 )) has been synthesized. This open glutaminyl-form has been compared with the cyclized pyro-form of (pyro)Glu-His-Pro(NH 2 ), thyrotropin releasing hormone (TRH). Gln-His-Pro(NH 2 ) is converted into (pyro)Glu-His-Pro(NH 2 ) under very mild conditions some of which are analogous to steps used in the isolation of TRH. Gln-His-Pro(NH 2 ) had up to five per cent of the activity of TRH although it is unknown whether this activity is intrinsic or due to contamination or conversion. There is no evidence that Gln-His-Pro(NH 2 ) is present in the hypothalamus, but if it were it could be a moiety of a larger molecule which is convertible into Gln- or (pyro)Glu-His-Pro(NH 2 ) or it could be a biosynthetic precursor or a metabolic transformation product of TRH. (Pyro)Glu-His-Pro(NH 2 ) shows the functions expected of the hormone.

Summary We probed the (immunochemical) relationship between the recently discovered growth hormone binding protein in human plasma and the growth hormone receptor using monoclonal and polyclonal antibodies raised against rabbit liver growth hormone receptor. The human binding protein was recognized by these antibodies; its immunological crossreactivity compared to the rabbit receptor was 1–2%. These data suggest a) that the binding protein and the receptor are structurally related and b) that rabbit and human growth hormone receptors share some but not all epitopes.

Competition for luteinizing hormone-releasing hormone (LH-RH) receptor sites by the inhibitory analog [D-Phe 2 , D-Trp 3 , D-Phe 6 ]-LH-RH and by the superactive stimulatory analog [D-Trp 6 ]-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.

Addition of gonadotropin releasing hormone to myo-[2-3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate within 30 sec of stimulation, followed by a rise in inositol diphosphate and inositol monophosphate. Inositol phosphate accumulation was enhanced up to 5-to-8-fold and was time-dependent between up to 15 min incubation without further increase beyond this time period. Without preincubation with LiCl2, there was no measurable increase of GnRH-induced inositol phosphate accumulation compared to controls. The presence of calcium in the incubation medium did not affect the increase of inositol phosphates. These data give evidence, that polyphosphoinositide breakdown may be an early step in the action of gonadotropin releasing hormone on gonadotropin secretion.

Pyroglutamylhistidylproline and histidylproline, reported metabolites of thyrotropin releasing hormone, were found to competitively inhibit purified rabbit lung angiotensin converting enzyme with K I values of 0.76 μM and 1.7 mM, respectively. Native thyrotropin releasing hormone and histidylprolinediketopiperazine at concentrations of 10 mM and 5 mM, respectively, had no effect on angiotensin converting enzyme activity. Neither the native hormone nor its deamidated derivative served as substrate for angiotensin converting enzyme.

Addition of thyrotropin-releasing hormone (TRH) to [ 3 h]-inositol pre-labelled GH 3 pituitary tumour cells suspended in medium containing 10mM lithium chloride led to a rapid diminution in cellular [ 3 H]-inositol and increase in [ 3 H]-inositol 1-phosphate (InauthorP 3 ), [ 3 H]-inositol bisphosphate (InsP 2 ) and [ 3 H]-inositol trisphosphate (InsP 3 ). In the presence of the benzodiazepine tranquillizer, chlordiazepoxide, the TRH concentration-response curves for these effects were shifted to the right in a parallel fashion. The K i for chlordiazepoxide in inhibiting all four responses was 1.5×10 −5 M. Chlordiazepoxide did not inhibit the small bombesin-induced rise in [ 3 H]-InauthorP. Another benzodiazepine, diazepam, was less active. The TRH-induced rise in cytosolic free calcium monitored in Quin-2-loaded GH 3 cells was also blocked by chlordiazepoxide in a competitive manner, while that induced by high K + -induced depolarisation was unaffected. It is suggested that chlordiazepoxide acts as a competitive antagonist at the level of the TRH receptor.

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly10-[Ala6]-LH-RH-ethylamide, des-Gly10-[D-Ala6]-LH-RH-ethylamide, des-Gly10-[α-aminoisobutyric acid6]-LH-RH-ethylamide, des-Gly10-[Phe5, D-Ala6]-LH-RH-ethylamide and des-Gly10-[Ile5, D-Ala6]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly10-[D-Ala6]-LH-RH-ethylamide and des-Gly10-[Phe5, D-Ala6]-LH-RH-ethylamide, were 50 times or more active than the original molecule.

Summary Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45 Ca 2+ from prelabeled anterior pituitary cells. Indomethacin (10 μM) and nordihydroguaiaretic acid (10 μM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45 Ca 2+ . Eicosatetraynoic acid (10 μM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45 Ca 2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45 Ca 2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.

Intraventricular administration of histidyl-proline diketopiperazine to rats produces a dose-dependent hypothermia at 4° or 24°, but not at 31°. At 4°, administration of thyrotropin releasing hormone elicits a dose-dependent hypothermia up to 0.1 μmole/kg which is not evoked at higher doses. At 24°, thyrotropin releasing hormone administration results in no change in core temperature, whereas it induces hyperthermia at 31°. At 4°, thyrotropin releasing hormone antagonizes and thyrotropin releasing hormone antiserum potentiates the hypothermic effect of histidyl-proline diketopiperazine, suggesting opposing actions of thyrotropin releasing hormone and histidyl-proline diketopiperazine on thermoregulation.

A new method is described for the measurement of hormone bound to membrane receptors. Antibodies specific for the C-terminal and N-terminal regions of parathyroid hormone were labelled with 125 I and incubated with renal membranes which had been previously incubated with unlabelled hormone. The uptake of hormone demonstrated pH and time dependence and was a saturable process. Treatment of the membranes with acid or heating to 100°C, or inactivation of the hormone with hydrogen peroxide, completely abolished detectable hormone uptake to the membranes.

We have previously described the binding of biologically active 125 I gonadotropin-releasing hormone to the 10,800 × g membrane fraction prepared from 7-day castrate adult female rat anterior pituitary glands. Specific binding with two equilibrium association constants (10 9 liters per mole and 10 5 liters per mole) was found and an equilibrium competitive binding radio-receptor assay established. In order to further characterize the gonadotropin-releasing hormone receptor, 20 synthetic analogs with known bioactivity were tested in the radioreceptor assay. In vivo biological activity correlated with high affinity receptor binding but not with low affinity binding. Inhibitory analogs with no in vivo biological activity and weak antagonistic properties did not bind, while in vivo active or superactive analogs bound to high affinity receptors. These findings suggest that the high affinity gonadotropin-releasing hormone receptor binds only biologically active gonadotropin-releasing hormone like peptides and that this binding may be the initial step in gonadotropin-releasing hormone actions at the pituitary level.

A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [3H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [3H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [3H]TRH, the 64 kDa protein cannot be demonstrated by [3H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells.

Five new analogs of LH-RH(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), ( Des-Gly-NH 2 10 , Pro-methylamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-propylamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-ethanolamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-pyrrolidineamide9)-LH-RH and ( Des-Gly-NH 2 10 , Pro-morpholineamide9)-LH-RH were synthesized and evaluated for the hormonal activity. The activities of these new analogs were compared with those of LH-RH and our previously reported highly potent analog, ( Des-Gly-NH 2 10 , Pro-ethylamide9)-LH-RH. The results demonstrate a significant contribution of the total chain-length of the hormone for the hormonal action.