Natural Peptide Hormone Research Articles

Relaxin as a biological marker and therapeutic target in heart failure

An important task of modern cardiology is the search and study of new cardiovascular biological markers that can help in the early diagnosis of heart diseases, serve as a tool to assess the effectiveness of treatment, and act as a prognostic marker and risk stratification criterion. This literature review aimed to consider relaxin (RLN) as a new diagnostic and prognostic cardiovascular biological marker. RLN is a natural peptide hormone with a molecular weight of approximately 6000 daltons. RLN2 is the main circulating form of RLN in the blood. Although human RLN2 was originally discovered as a hormone mainly secreted by the corpus luteum of the ovary, it is also synthesized in various tissues in non-pregnant women and men and is considered one of the most pleiotropic hormones of the human body, which perform various activities beyond reproduction.
 The availability of serelaxin (a recombinant molecule identical to human RLN2) has made it possible to study the effects of RLN2 on the cardiovascular system, kidneys, liver, and brain and to evaluate it in several randomized placebo-controlled clinical trials. RLN2 exerts many cardioprotective effects on the heart and vessels and has been proposed as a therapeutic target for cardiovascular diseases such as heart failure, atrial fibrillation, coronary heart disease, myocardial infarction, and arterial hypertension. Through the activation of its cognate receptor and subsequent flow of several molecular signaling pathways in the cardiovascular system, RLN2 can induce vasodilation and angiogenesis, increase arterial compliance and cardiac output, reduce vascular resistance, exert antifibrotic effects through regulation and remodeling of extracellular matrix turnover, reduce inflammation and apoptosis, inhibit oxidative stress, induce chronotropic and inotropic effects, and inhibit ventricular and atrial ectopic activity.
 Further studies are needed to demonstrate the potential use of RLN as an additional laboratory tool for diagnosis, risk stratification, and prediction of cardiovascular events in patients with heart failure. The effects of relaxin on morbidity and mortality in patients with heart failure have yet to be evaluated in more detail.

Update on Preclinical Development and Clinical Translation of Cholecystokinin-2 Receptor Targeting Radiopharmaceuticals.

Simple SummaryPeptide analogs, derived from the natural peptide hormone gastrin, are promising candidates for improving the visualization and treatment of tumors. Gastrin specifically binds to the cholecystokinin-2 receptor, a G-protein-coupled receptor expressed on the cell surface of different tumors. This enables specific targeting of tumor cells using gastrin analogs, labeled with radioisotopes. The receptor is expressed at high incidence in medullary thyroid carcinoma, a rare form of thyroid cancer lacking effective treatments at an advanced stage. Different radiolabeled gastrin analogs as well as nonpeptidic compounds targeting CCK2R have been developed. Specific modifications have been introduced in order to safely deliver the radiation to the tumor site. In this review, recent strategies applied to improve the targeting properties are described. These developments enabled the introduction of new radiolabeled peptide analogs for imaging and therapy in cancer patients. In addition to highlighting the current clinical trials, the perspectives for future applications are given.The cholecystokinin-2 receptor (CCK2R) has been a target of interest for molecular imaging and targeted radionuclide therapy for two decades. However, so far CCK2R targeted imaging and therapy has not been introduced in clinical practice. Within this review the recent radiopharmaceutical development of CCK2R targeting compounds and the ongoing clinical trials are presented. Currently, new gastrin derivatives as well as nonpeptidic substances are being developed to improve the properties for clinical use. A team of specialists from the field of radiopharmacy and nuclear medicine reviewed the available literature and summarized their own experiences in the development and clinical testing of CCK2R targeting radiopharmaceuticals. The recent clinical trials with novel radiolabeled minigastrin analogs demonstrate the potential for both applications, imaging as well as targeted radiotherapy, and reinforce the clinical applicability within a theranostic concept. The intense efforts in optimizing CCK2R targeting radiopharmaceuticals has led to new substances for clinical use, as shown in first imaging studies in patients with advanced medullary thyroid cancer. The first clinical results suggest that the wider clinical implication of CCK2R-targeted radiopharmaceuticals is reasonable.

Study Protocol of the PreFiPS Study: Prevention of Postoperative Pancreatic Fistula by Somatostatin Compared With Octreotide, a Prospective Randomized Controlled Trial.

Background: Pancreatic fistula (PF), i. e., a failure of the pancreatic anastomosis or closure of the remnant pancreas after distal pancreatectomy, is one of the most feared complications after pancreatic surgery. PF is also one of the most common complications after pancreatic surgery, occurring in about 30% of patients. Prevention of a PF is still a major challenge for surgeons, and various technical and pharmacological interventions have been investigated, with conflicting results. Pancreatic exocrine secretion has been proposed as one of the mechanisms by which PF occurs. Pharmacological prevention using somatostatin or its analogs to inhibit pancreatic exocrine secretion has shown promising results. We can hypothesize that continuous intravenous infusion of somatostatin-14, the natural peptide hormone, associated with 10–50 times stronger affinity with all somatostatin receptor compared with somatostatin analogs, will be associated with an improved PF prevention.Methods: A French comparative randomized open multicentric study comparing somatostatin vs. octreotide in adult patients undergoing pancreaticoduodenectomy (PD) or distal pancreatectomy with or without splenectomy. Patients with neoadjuvant radiation therapy and/or neoadjuvant chemotherapy within 4 weeks before surgery are excluded from the study. The main objective of this study is to compare 90-day grade B or C postoperative PF as defined by the last ISGPF (International Study Group on Pancreatic Fistula) classification between patients who receive perioperative somatostatin and octreotide. In addition, we analyze overall length of stay, readmission rate, cost-effectiveness, and postoperative quality of life after pancreatic surgery in patients undergoing PD.Conclusion: The PreFiPS study aims to evaluate somatostatin vs. octreotide for the prevention of postoperative PF.

Mechanisms and Strategies Shaping Plant Peptide Hormones.

Plant genomes encode a variety of short peptides acting as signaling molecules. Since the discovery of tomato systemin, a myriad of peptide signals, ranging in size, structure and modifications, have been found in plants. Moreover, new peptides are still being identified. Surprisingly, non-plant organisms, especially pathogens, also produce peptides which exert hormonal activities against host plants by hijacking their endogenous reception systems. In this review, we focus on short secretory peptides ranging from five to 20 amino acids. We first summarize recent advances in understanding relationships between the bioactivities and structures of plant peptide hormones. Subsequently, we introduce the topic of peptides produced by non-plant organisms. Lastly, we describe artificial peptides synthesized in laboratories, which possess intriguing bioactive properties beyond those of natural peptide hormones.

Peptide hormones and lipopeptides: from self-assembly to therapeutic applications.

This review describes the properties and activities of lipopeptides and peptide hormones and how the lipidation of peptide hormones could potentially produce therapeutic agents combating some of the most prevalent diseases and conditions. The self‐assembly of these types of molecules is outlined, and how this can impact on bioactivity. Peptide hormones specific to the uptake of food and produced in the gastrointestinal tract are discussed in detail. The advantages of lipidated peptide hormones over natural peptide hormones are summarised, in terms of stability and renal clearance, with potential application as therapeutic agents. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Histidine 379 of Human Laeverin/Aminopeptidase Q, a Nonconserved Residue within the Exopeptidase Motif, Defines Its Distinctive Enzymatic Properties

Human laeverin/aminopeptidase Q (LVRN/APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of human extravillous trophoblasts. Multiple sequence alignment of human M1 aminopeptidase revealed that the first Gly residue within the conserved exopeptidase motif of the M1 family, GXMEN motif, is uniquely substituted for His in human LVRN/APQ. In this study, we evaluated the roles of nonconserved His(379), comprising the exopeptidase motif in the enzymatic properties of human LVRN/APQ. We revealed that the substitution of His(379) with Gly caused significant changes in substrate specificity both toward fluorogenic substrates and natural peptide hormones. In addition, the susceptibilities of bestatin, a sensitive inhibitor for human LVRN/APQ, and natural inhibitory peptides were decreased in the H379G mutant. A molecular model suggested a conformational difference between wild-type and H379G human LVRN/APQs. These results indicate that His(379) of the enzyme plays essential roles in its distinctive enzymatic properties and contributes to maintaining the appropriate structure of the catalytic cavity of the enzyme. Our data may bring new insight into the biological significance of the unique exopeptidase motif of LVRN/APQ obtained during the evolution of primates.

Urotensin II and renal function in the rat

Urotensin II (UII) is a potent vasoactive hormone in mammals. However, despite its well-known effects on epithelial sodium transport in fish, little is known about its actions on the mammalian kidney. The aim of this study was to determine the effects of UII on renal function in the rat. Using standard clearance methods, the effects of rUII and the rat UII receptor (UT) antagonist, urantide, were studied. UII was measured in plasma and urine by radioimmunoassay. UII and UT were localized in the kidney by immunohistochemistry and mRNA expression quantified. Rat urinary [UII] was 1,650-fold higher than that in plasma. Immunoreactive-UII was localized to the proximal tubules, outer and inner medullary collecting ducts (IMCD); UT receptor was identified in glomerular arterioles, thin ascending limbs, and IMCD. UII and UT mRNA expression was greater in the medulla; expression was higher still in spontaneously hypertensive rats (SHRs) associated with raised plasma (UII). Injection of rUII induced reductions in glomerular filtration rate (GFR), urine flow, and sodium excretion. Urantide infusion resulted in increases in these variables. Endogenous UII appears to contribute to the regulation of GFR and renal sodium and water handling in the rat. While hemodynamic changes predominate, we cannot rule out the possibility of a direct tubular action of UII. Increased expression of UII and UT in the SHR suggests that UII plays a role in the pathophysiology of cardiovascular disease.

Diabetes mellitus and retinopathy

The role of somatostatin and growth hormone in eye diseases recently became a matter of interest because of its link with proliferative diabetic retinopathy. In diabetic patients the pathologic proliferation of blood vessels as a result of retinal ischemia is a major cause of blindness. The hypoxic portions of the retina release angiogenic factors, stimulating neovascularization. Somatostatin is a natural peptide hormone that affects the release of a number of other hormones, such as growth hormone, glucagon, insulin and gastrin. The somatostatin analog promises to be safe and effective treatment for severe diabetic retinopathy. This compound has been shown to block the local and systemic production of insulin-like growth factor 1 and growth hormone, which promote the angiogenesis and endothelial cell proliferation associated with proliferative retinopathy. Several studies have confirmed that using somatostatin analogs to block insulin-like growth factor 1 production is effective in reducing neovascularization and preventing disease progression to proliferative stage of diabetic retinopathy. Long-acting somatostatin analogs are currently being tested for the treatment of diabetic retinopathy. The development of somatostatin analogs with increased selectivity for receptor subtypes will provide improved outcomes in the management of patients with diabetic retinopathy.

Expression of the bovine growth hormone alters the root morphology in transgenic tobacco plants.

The bovine growth hormone (bGH) is a natural peptide hormone that controls the differentiation, growth and metabolism, and is produced in the pituitary gland of cows. For the production of bGH from plants, two different bgh clones, of which the pGAbGH1 contaions only mature peptide sequences and the pGAbGH15 contains signal sequences and the first intron, as well as mature peptide sequences, were used. Those bghs under the control of the CaMV 35S promoter and NOS terminator were introduced to tobacco plants via Agrobacterium tumefaciens-mediated transformation. By PCR analyses using bgh and nptII specific primers, 17 and 21 putative transformants were respectively selected from pGAbGH1- and pGAbGH15-transformed tobacco plants. Northern blot analysis showed that the most of the transgenic lines expressed the bgh mRNA. Western blot analysis revealed that the pGAbGH1-transformed tobaccos produced recombinant bGH, but pGAbGH15-transformed ones did not produce the protein. Interestingly, some morphological changes were observed in the roots of transgenic tobacco plants. The transgenic tobacco plants had thick and short roots containing few root hairs in contrast to the non-transformed wild type plants.

The Cyclo-β-Tetrapeptide (β-HPhe-β-HThr-β-HLys-β-HTrp): Synthesis, NMR Structure in Methanol Solution, and Affinity for Human Somatostatin Receptors

The known solid-state structure (Fig. 1, top) of cyclo(β-HAla)4 was used to model the structure of the title compound 1 as a prospective somatostatin mimic (Fig. 1, bottom). The synthesis started with the N-protected natural amino acids Boc-Phe-OH, Boc-Trp-OH, Boc-Lys(2-Cl-Z)-OH, and Boc-Thr(OBn)-OH, which were homologated to the corresponding β-amino-acid derivatives (Scheme 1) and coupled to the β-tetrapeptide Boc-β-HTrp-β-HPhe-β-HThr(OBn)-β-HLys(2-Cl-Z)-OMe (16); the (N-Me)-β-HThr-(N-Me)-β-HPhe analog 17 was also prepared. C- and N-terminal deprotection and cyclization through the pentafluorophenyl ester gave the insoluble β-tetrapeptide with protected Thr and Lys side chains (18). Solubilization and debenzylation could only be effected in LiCl-containing THF (ca. 10% yield; with ca. 55% recovery). HPLC Purification provided a sample of the title compound 1, the structure of which, as determined by NMR-spectroscopy (Fig. 2, left) was drastically different from the `theoretical' model (Fig. 1). There is a transannular H-bond dividing the macrocyclic 16-membered ring, thus forming a ten- and a twelve-membered H-bonded ring, the former mimicking, or actually being superimposable on, an α-peptidic so-called β-turn. Still, the four side chains occupy equatorial positions on the ring, as planned, albeit with somewhat different geometry as compared to the `original'. The cyclo-β-tetrapeptide has micromolar affinities to the human somatostatin receptors (hsst 1 – 5). Thus, we have demonstrated for the first time that it is possible to mimic a natural peptide hormone with a small β-peptide. Furthermore, we have discovered a simple way to construct the ubiquitous β-turn motif with β-peptides (which are known to be stable to mammalian peptidases).

Synthesis and Biological Evaluation of a Cyclo-β-tetrapeptide as a Somatostatin Analogue.

Short β-peptides can mimic natural peptide hormones, as has been shown with a cyclo-β-tetrapeptide (1) that displays micromolar affinity to human somatostatin receptors. β-Peptides are thus a promising new class of peptidomimetics with potential high bioavailability due to their excellent resistance against proteases.

Radioiodinated endothelin-1: a radiotracer for imaging endothelin receptor distribution and occupancy

Endothelin (ET) is one of the most potent vasoconstrictors known. Recently, ET has been implicated in various diseases, e.g., acute renal failure and congestive heart failure, which present the possibility of treating such diseases with endothelin receptor antagonists. However, establishing the dosages for these antagonists may be difficult because no convenient physiologic indicator of action exists, and because of complexities in receptor function. Two receptor subtypes have been identified for which selective antagonists have been reported (e.g., BQ-123 for the ET A receptor and BQ-788 for the ET B receptor). Of the three natural peptide hormones (ET-1, ET-2, and ET-3), ET-1 exhibits high affinity for both subtypes of receptor. Using the selective peptide antagonists, and a nonpeptide antagonist with relatively balanced affinity for the two subtypes (L-749,329), we have characterized the interactions of [ 125I]ET-1 with its receptors in vivo (in rat). BQ-123, BQ-788, and L-749,329 inhibited binding consistent with binding to a single receptor site. However, the sum of inhibition by the selective antagonist was greater than 100% (as defined by inhibition with L-749,329), which suggests (a) lower in vivo selectivity than determined in vitro and/or (b) receptor subtype interactions. The latter explanation is supported, in part, by in vitro autoradiographic studies as well as studies in isolated tissues and cells. We synthesized ET-1 labeled with I-123 and obtained images of receptor distribution in both rat and rhesus monkey and have demonstrated our ability to visualize, via planar, noninvasive imaging, the occupancy of endothelin receptor by antagonists in both kidney and lung. [ 123I]ET-1 can therefore be used to determine clinical dosages of antagonist needed for receptor saturation.

Glucagon-like peptide 1 (GLP-1): a potent gut hormone with a possible therapeutic perspective.

Glucagon-like peptide 1 (GLP-1) is a physiological incretin hormone from the lower gastrointestinal tract, partially explaining the augmented insulin response after oral compared to intravenous glucose administration in normal humans. In addition, GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro)insulin biosynthesis, and reduces food intake upon intracerebroventricular administration in animals. Therefore, GLP-1 offers some interesting perspective for the treatment of type 2, and perhaps also for type 1 diabetic patients. The other incretin hormone, gastric inhibitory polypeptide (GIP), has lost almost all its activity in type-2 diabetic patients. In contrast, GLP-1 glucose-dependently stimulates insulin secretion in type-2 diabetic patients and exogenous administration of GLP-1 ([7-37] or [7-36 amide]) in doses elevating plasma concentrations to approximately three to four times physiological postprandial levels fully normalizes fasting hyperglycaemia and reduces postprandial glycaemic increments. Due to rapid proteolytic cleavage, which results in an inactive or even antagonistic fragment. GLP-1 [9-36 amide], and to rapid elimination, the half-life of GLP-1 is too short to maintain therapeutic plasma levels for sufficient periods by subcutaneous injections of the natural peptide hormone. Current research aims to characterize GLP-1 analogues with more suitable pharmacokinetic properties than the original peptide. Given the large amount of GLP-1 present in L cells, it also appears worthwhile to search for more agents that could 'mobilize' this endogenous pool of GLP-1.

Analytical strategies in the structural characterization of elcatonin.

Elcatonin is a synthetic peptide of 32 amino acid residues, that differs from natural peptide hormone (eel calcitonin) in that the 1 and 7 cystine residues are replaced with alpha-amino suberic acid (Asu). Elcatonin is pharmacologically important, since it inhibits osteoclastic bone reserption and induces calcium uptake from body fluids. It is also used for the treatment of Page's disease and hypercalcemic conditions. Until now the structural characterization of elcatonin has been obtained by proteolytic digestion followed by high performance liquid chromatographic (HPLC) analysis of the peptide fragments. Capillary electrophoresis and fast-atom bombardment have also been employed. This work describes the results obtained when a liquid chromatograph, coupled to mass spectrometer using electrospray ionization (LC/ESI-MS) was applied to elcatonin analysis. After digestion with trypsin, the resulting peptides were separated by HPLC with 'on-line' UV detection, and directly injected into the ESI source. The molecular weights of all the fragments were detected, and the sequences of two of them were determined by collisionally induced dissociation in the ESI source. To confirm these 'on-line' results, the 'off-line' approach was also applied. In this case, the fragments from tryptic digestion were isolated by preparative HPLC, concentrated and analyzed by direct infusion into the ESI-MS system. Then, different elcatonin digests obtained using other proteases, e.g. protease V8 and clostripain, were analyzed by direct infusion, and these results combined with those achieved by the 'on-line' analysis allowed us to obtain the entire mapping of elcatonin.

Analytical strategies in the structural characterization of elcatonin

Elcatonin is a synthetic peptide of 32 amino acid residues, that differs from natural peptide hormone (eel calcitonin) in that the 1 and 7 cystine residues are replaced with alpha-amino suberic acid (Asu). Elcatonin is pharmacologically important, since it inhibits osteoclastic bone reserption and induces calcium uptake from body fluids. It is also used for the treatment of Page's disease and hypercalcemic conditions. Until now the structural characterization of elcatonin has been obtained by proteolytic digestion followed by high performance liquid chromatographic (HPLC) analysis of the peptide fragments. Capillary electrophoresis and fast-atom bombardment have also been employed. This work describes the results obtained when a liquid chromatograph, coupled to mass spectrometer using electrospray ionization (LC/ESI-MS) was applied to elcatonin analysis. After digestion with trypsin, the resulting peptides were separated by HPLC with ‘on-line’ UV detection, and directly injected into the ESI source. The molecular weights of all the fragments were detected, and the sequences of two of them were determined by collisionally induced dissociation in the ESI source. To confirm these ‘on-line’results, the ‘off-line’ approach was also applied. In this case, the fragments from tryptic digestion were isolated by preparative HPLC, concentrated and analyzed by direct infusion into the ESI-MS system. Then, different elcatonin digests obtained using other proteases, e.g. protease V8 and clostripain, were analyzed by direct infusion, and these results combined with those achieved by the ‘on-line’ analysis allowed us to obtain the entire mapping of elcatonin. © 1997 John Wiley & Sons, Ltd.

A new approach to receptor ligand design: synthesis and conformation of a new class of potent and highly selective μ opioid antagonists utilizing tetrahydroisoouinoline carroxylic acid

Investigations of the physiological functions of opioid receptors (μ,δ,κ and others) require potent and selective receptor ligands. Conformational constraint provides a useful approach to increase receptor selectivity of flexible peptides. This approach reduces the set of low energy conformations accessible for the ligand and thus can provide insight into topologica1 features that may be responsible for high affinity to a particular receptor subtype. Using this approach, we describe a new class of potent and selective μ opioid receptor antagonists and demonstrate a new approach for the design of receptor specific ligands by which a low affinity, “non-physiological” activity of a natural peptide hormone is converted to a high potency, receptor selective ligand for that receptor, and, at the same time, eliminates the activity at the natural receptors for the peptide. Recently we reported the design and synthesis of a new class of μ opioid receptor selective antagonists, of which ▪(CTP) was found to be among the most potent and selective, and ▪(PCTP) exhibited a sharp decrease of affinity for μ receptors ( 100 foTd) and a modest increase ( 3 fold) in affinity for δ receptors. We now present 1h NMR evidence which suggests a more folded conformation for the latter compound. This result led to the design of further constrained analogues in which a methylene bridge is inserted between the α-amino group and the 2'position of the aromatic ring of n -phe 1 in CTP. This analogue ▪(TCTP) was found to be the most μ vs. δ receptor selective ligand known (> 9000 fold selective), with very litt1e somatostatin-like activity. NMR investigations have revealed that the side chain of D -Tic residue exists exclusively in a g - conformation. Disconnection of this methylene bridge via synthesis of ▪ gave an analogue that exhibited low affinity for the μ opioid receptor and greatly reduced selectivity. NMR investigations have uncovered a large participation of g + and trans side chain conformations for the aromatic ring in the D -N-MePhe residue, and a more folded overall conformation. These results illustrate how constraint of side chain moieties of critical amino acid residues to a specific or “biased” conformation can provide important insights into the topological requirements for peptide-receptor interactions and can contribute to design of ligands for receptor mapping.

Biological and physical properties of a beta-endorphin analog containing only D-amino acids in the amphiphilic helical segment 13-31.

Our approach to the modeling of beta-endorphin has been based on the proposal that three basic structural units can be distinguished in the natural peptide hormone: a highly specific opiate recognition sequence at the N terminus (residues 1-5) connected via a hydrophilic link (residues 6-12) to a potential amphiphilic helix in the C-terminal residues 13-31. Our previous studies showed the validity of this approach and have demonstrated the importance of the amphiphilic helical structure in the C terminus of beta-endorphin. The present model, peptide 5, has been designed in order to evaluate further the requirements of the amphiphilic secondary structure as well as to determine the importance of this basic structural element as compared to more specific structural features which might occur in the C-terminal segment. For these reasons, peptide 5 retains the three structural units previously postulated for beta-endorphin; the major difference with regard to previous models is that the whole C-terminal segment, residues 13-31, has been built using only D-amino acids. In aqueous buffered solutions as well as in 2,2,2-trifluoroethanol-containing solutions, the CD spectra of peptide 5 show the presence of a considerable amount of left-handed helical structure. Enzymatic degradation studies employing rat brain homogenate indicate that peptide 5 is stable in this milieu. In delta- and mu-opiate receptor-binding assays, peptide 5 shows a slightly higher affinity than beta-endorphin for both receptors while retaining the same delta/mu selectivity. In opiate assays on the guinea pig ileum, the potency of peptide 5 is twice that of beta-endorphin. In the rat vas deferens assay, which is very specific for beta-endorphin, peptide 5 displays mixed agonist-antagonist activity. Most remarkably, peptide 5 displays a potent opiate analgesic effect when injected intracerebroventricularly into mice. At equal doses, the analgesic effect of peptide 5 is less than that of beta-endorphin (10-15%) but longer lasting. In conjunction with our previous model studies, these results clearly demonstrate that the amphiphilic helical structure in the C terminus of beta-endorphin is of predominant importance with regard to activity in rat vas deferens and analgesic assays. The similarity between the in vitro and in vivo opiate activities of beta-endorphin and peptide 5, when compared to the drastic change in chirality in the latter model, demonstrates that even a left-handed amphiphilic helix formed by D-amino acids can function satisfactorily as a structural unit in a beta-endorphin-like peptide.

Specificity of response of intestinal ion transport systems to a pair of natural peptide hormone analogs: Somatostatin and urotensin II

Specificity of two intestinal ion transport systems toward the natural peptide hormone analogs somatostatin and urotensin II has been demonstrated by electrophysiological and radiotracer studies in vitro. Somatostatin inhibits active Cl secretion across the theophylline-treated rat colon but urotensin II, a dodecapeptide somatostatin analog from the teleost caudal neurosecretory system, is without effect. Conversely, urotensin II stimulates active Na and Cl absorption across the posterior intestine of the 5% seawater-adapted gody, Gillichthys mirabilis, but somatostatin is ineffective. From these and others' studies, it appears that in both systems, increased net absorption results from increased mucosal-to-serosal unidirectional ion fluxes. Based on structure-activity relationships in these and other system, it is speculated that the difference in amino acid residues at position 4 (somatostatin—Lys, urotensin II—Ala) may contribute to the observed specificity.

13C-nuclear magnetic resonance study of [85% 13C-enriched proline]thyrotropin releasing factor: 13C-13C vicinal coupling constants and conformation of the proline residue.

To understand fully interactions between peptides and cellular receptors, peptide side chain conformation must be defined. In many cases the complexity of proton nuclear magnetic resonance (NMR) prevents this but the present work demonstrates this problem can be solved by using 13C enrichment. Selective 13C enrichment of a natural peptide hormone has been achieved by preparing [85% 13C-enriched proline]thyrotropin releasing factor which was examined by 13C NMR spectroscopy at various pH values. Because of the 13C enrichment, one-bonded and three-bonded (vicinal) 13C-13C coupling constants have been determined. The latter vary from 0 to 5 Hz and show bond angle dependence. These data indicate that in this hormone the pyrrolidine ring is not free but fixed in the Cgamma-endo puckered conformation. It has also been possible to assign chemical shift values for a second order 13C NMR spectrum.

4-Threonine analogues of neurohypophysial hormones withh selectively enhanced oxytocin-like activities.

SUMMARY The 4-threonine analogues of oxytocin and of mesotocin and isotocin were prepared by solid-phase synthesis. [4-Threonine]-oxytocin is about twice as active as oxytocin in rat uterus assays in vitro and in vivo and about three times as active in fowl vasodepressor assays. It is slightly more active than oxytocin in rat or rabbit milk-ejection assays. When infused intravenously into water-loaded rats it causes much less depression of diuresis than does an equal dose of oxytocin. [4-Threonine]-oxytocin has much less vasopressor activity than oxytocin. [4-Threonine]-mesotocin also shows enhanced oxytocin-like properties. Its oxytocic activity is equal to or greater than that of oxytocin and its fowl vasodepressor potency is about the same as that of [4-threonine]-oxytocin, 1500 u./mg. It also has less antidiuretic and vasopressor activities than mesotocin. Thus 4-threonine analogues, containing nothing but common l-amino acids, appear to have more of the specific oxytocin-like properties and less of the vasopressin-like properties than do oxytocin or mesotocin. Thus they may be considered improvements on the natural hormones. In this respect they are unique among the reported synthetic analogues of natural peptide hormones. Substitution of 4-threonine in the weakly-active analogue [3-leucine]-oxytocin also increases its oxytocic and fowl vasodepressor activities. Thus a threonine in the 4-position appears to endow oxytocin-like peptides with greater specific activities than do the amino acids that occur naturally in this position, glutamine and serine. These observations may be of interest when considered (a) from an evolutionary viewpoint, (b) in attempting to interpret relations between molecular structures and biological activities, and (c) as describing peptides with more of the desired properties of oxytocin and less of the undesired properties which might have therapeutic advantages over the natural hormone.